EXCHANGE 


THE  BACTERIA  OF  NEBRASKA  SOIL 

WITH  SPECIAL  REFERENCE 

TO   THE  FIXATION  OF  NITROGEN,  AMMONIFICATION 

DENITRIFICATION  IN  NON-PROTEIN  MEDIA, 

INCLUDING  OBSERVATIONS  ON  THE 

REDUCTION  OF    NITRATES  BY 

SOIL  BACTERIA  IN 

GENERAL 


BY 

JOHN  J.  PUTNAM 


OF    THE 

UNIVERSITY 


SUBMITTED  TO  THE 

GRADUATE  COLLEGE 

OF 

THE  UNIVERSITY  OF  NEBRASKA 

IN  CANDIDACY  FOR  THE  DEGREE 
DOCTOR  OF  PHILOSOPHY 


FEBRUARY  20,  1913 


1913 

THE   WOODRUFF    PRESS 
LINCOLN,  NEB. 


THE  BACTERIA  OF  NEBRASKA  SOIL 

WITH  SPECIAL  REFERENCE 

TO   THE  FIXATION  OF   NITROGEN,  AMMONIFICATION 

DENITRIFICATION  IN  NON-PROTEIN  MEDIA, 

INCLUDING  OBSERVATIONS  ON  THE 

REDUCTION  OF    NITRATES  BY 

SOIL  BACTERIA  IN 

GENERAL 


BY 

JOHN  J.  PUTNAM 


SUBMITTED  TO  THE 

GRADUATE  COLLEGE 

OF 

THE  UNIVERSITY  OF  NEBRASKA 

IN  CANDIDACY  FOR  THE  DEGREE 
DOCTOR  OF  PHILOSOPHY 


FEBRUARY  20.  1913 


1913 

THE  WOODRUFF    PRESS 
LINCOLN,  NEB. 


THE  BACTERIA  OF  NEBRASKA  SOIL  WITH  SPECIAL 

REFERENCE  TO  THE  FIXATION  OF  NITROGEN, 

AMMONIFICATION,  DENITRIFICATION  IN 

NON-PROTEIN   MEDIA   INCLUDING 

OBSERVATIONS  ON  THE  REDUCTION  OF 

NITRATES  BY  SOIL  BACTERIA 

IN  GENERAL 


HISTORICAL 

This  work  was  undertaken  with  the  idea  of  ascertaining 
if  possible,  some  of  the  many  chemical  changes  taking  place 
through  the  action  of  bacteria  indigenous  to  Nebraska  soil. 

The  fixation  of  nitrogen  was  first  observed  by  M.  Berthelot 
in  1885.  He  subsequently  was  able  to  prove  that  this  pheno- 
menon is  not  brought  about  exclusively  by  a  purely  chemical 
process,  but  is  due  to  the  activity  of  micro-organisms.  The 
discovery  of  an  anaerobic  organism  by  S.  Winogradsky  in 
1893,  Clostridium  pasteurianum,  which  he  found  fixed  from 
2.5  to  3  mg  of  nitrogen  per  gram  of  dextrose  consumed,  marked 
the  first  advance  along  this  important  line.  Recent  observers 
have  added  a  few  organisms  to  the  list,  Beyerinck,  Lohnis 
and  Lipman  having  labored  successfully  in  this  field. 

In  1887  Schlossing  and  Muntz  hazarded  the  opinion  that 
the  formation  of  nitrate  within  the  soil  is  due  to  the  vital 
activity  of  soil  bacteria,  and  in  a  subsequent  communication 
these  two  workers  detailed  some  of  the  conditions  requisite 
for  the  inception  and  course  of  nitrification.  Much  opposition 
developed  from  the  advocates  of  the  chemical  theory.  A  re- 
examination  of  the  comprehensive  work  of  H.  Plath  by  Lon- 
dalt,  who  undertook  the  task  in  consequence  of  an  objection 
raised  by  B.  Frank,  led  to  a  complete  confirmation  of  Plath's 
discoveries  in  all  particulars.  It  was  thus  ascertained  in  1888, 
by  the  exclusion  method,  that  in  the  oxidation  process  now 
under  our  notice  the  role  of  oxygen-carrier  is  played  by  living 
organisms,  and  that  nitrification  consequently  is  a  physiological 
process.  The  discovery  and  closer  investigation  of  these  un- 
known organisms  was  shortly  afterwards  effected  by  S.  Wino- 


gradsky,  who  isolated  them  in  pure  culture.  Of  great  im- 
portance is  the  fact  determined  by  Winogradsky  that  the 
numerous  species  of  the  group  of  nitrifying  bacteria  may  be 
classified  into  two  sharply  divided  sub-groups:  Nitroso- 
bacteria  and  Nitro-bacteria.  The  nitroso-bacteria  oxidize 
ammonia  to  nitrous  acid,  while  the  nitro-bacteria  lack  the 
faculty  of  attacking  ammonia,  but  perform  the  important 
task  of  converting  nitrous  acid  into  nitric  acid. 

We  are  indebted  to  E.  Marchel  for  proving  that  the 
faculty  of  eliminating  ammonia  from  albuminoids  is  common 
to  many  fungi.  The  potency  of  the  different  species  was 
found  by  him  to  vary,  the  largest  quantity  being  produced  by 
Bacillus  mycoides. 

The  first  researches  along  the  line  of  denitrification  were 
undertaken  by  Jules  Reiset  in  1854  and  1855.  He  asserted 
that  free  nitrogen  was  always  evolved  during  the  decomposi- 
tion of  manure.  Denitrification  in  arable  soil  was  first  noticed 
by  Gappelsroder  in  1862,  and  was  long  regarded  as  a  purely 
chemical  process.  The  first  reference  to  the  agency  of  bacteria 
in  this  decomposition  was  made  by  E.  Mensel  in  1875,  and  the 
earliest  pure  cultures  of  such  organisms  were  obtained  by  U. 
Gay  on  and  G.  Dupetit  in  1882.  In  succeeding  years  a  large 
number  of  species,  all  capable  of  reducing  nitrates,  was  made 
known.  In  1888,  P.  Frankland  was  able  to  associate  with  the 
group  in  question  17  out  of  32  species,  and  R.  Warington  16 
out  of  25  species  examined.  A.  Maassen,  in  1902,  found  that 
out  of  109  species,  85  were  able  to  perform  this  function. 
But  few  of  the  organisms  which  have  been  observed  to  reduce 
nitrates  to  nitrites  in  pure  culture,  are  able  to  continue  the 
reduction  to  the  liberation  of  free  nitrogen. 


For  an  inquiry  into  the  various  functions  performed  by  soil 
bacteria  in  general  and  with  reference  to  the  factors  concerned  in 
the  fixation  of  nitrogen  by  azotobacter,  ammonification,  reduc- 
tion of  nitrates  and  denitrification,  in  particular,seventy  samples 
of  soil  were  taken.  These  soils  comprise  perhaps  all  of  the  various 
types  within  our  borders,  with  the  possible  exception  of  the  alkali 
tracts  which  are  interspersed  over  the  western  half  of  our 
state.  Locations  were  made  not  with  special  reference  to  any 
favored  locality  or  type  of  soil,  but  rather  that  the  samples 


should  be  fairly  representative  of  the  whole  state.  These 
samples  were  taken  in  tubes  which  were  constructed  especially 
for  our  purpose,  and  are  of  steel  bicycle  tubing,  eight  inches 
long  and  one  inch  in  diameter.  Whenever  possible,  the  earth 
was  removed  in  a  crust  of  an  inch  or  less  in  thickness,  and 
the  tube  forced  into  the  ground  beneath,  to  a  depth  of  four  or 
five  inches.  About  one  inch  of  dirt  was  removed  from  the 
tube  with  a  sterile  knife,  and  the  cotton  plug  readjusted.  On 
being  returned  to  the  laboratory  the  samples  were  transferred 
to  sterile  four  ounce  salt-mouth  bottles,  thoroughly  mixed, 
and  soil  taken  therefrom  as  desired.  Experience  has  abund- 
antly proven  that  the  whole  process  of  sampling,  transfer,  etc., 
can  be  performed  with  such  exactness  that  no  contamination 
takes  place. 

The  following  list  shows  the  character  of  each  of  the  soils 
investigated : 


1 
2 
3 
4 
5 
6 

7 
8 
9 

10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 


Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Silt  loam 
Silt  loam 
Silt  loam 
Fine  sand  loam 
Fine  sand  loam 
Silt  loam 
Silt  loam 
Silt  loam 
Silt  loam 


25  Silt  loam  49 

26  Silt  loam  50 

27  Loam  51 

28  Loam  52 

29  Loam  53 

30  Loam  54 

31  Fine  sand  loam  55 

32  Loam  56 

33  Loam  57 

34  Loam  58 

35  Loam  59 

36  Loam  60 

37  Loam  61 

38  Loam  62 

39  Loam  63 

40  Silt  loam  64 

41  Loam  65 

42  Loam  66 

43  Loam  67 

44  Silt  loam  68 

45  Fine  sand  loam  69 

46  Fine  sand  loam  70 

47  Silt  loam 

48  Muck 


Muck 
Loam 
Loam 
Silt  loam 
Loam 
Clay  loam 
Silt  loam 
Silt  loam 
Calcareus  clay 
Fine  sand  loam 
Silt  loam 
Fine  sand  loam 
Fine  sand  loam 
Gravel 

Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Fine  sand  loam 
Calcareus  loam 
Fine  sand  loam 
Fine  sand  loam 


No  more  samples  were  taken  at  one  time  than  could  be 
handled  with  promptness,  therefore  necessitating  several  trips 
over  the  state. 


In  order  to  approximate  the  number  of  bacteria  within  the 
soil,  the  seventy  samples  were  plated  both  on  nutrient  agar, 
and  Ashby's  medium  to  which  was  added  fifteen  grams  of  agar- 
agar  per  liter. 

Nutrient  medium  recommended  by  Ashby  for  fixation  of 
nitrogen  by  azotobacter: 

Mannite 20.00  grams 

Di-potassium  phosphate 0.20  gram 

Magnesium  sulphate 0.20  gram 

Sodium  chloride 0.20  gram 

Calcium  sulphate 0.10  gram 

Calcium  carbonate 5.00  grams 

Distilled  water 1000.00  grams 

A  Kjeldahl  determination  disclosed  the  fact  that  good 
agar-agar  contained  0.16%  of  nitrogen,  or  a  little  below  the 
average  of  good  soil.  Therefore,  each  plate  of  the  Ashby 
medium  contained  approximately  .25  mg  of  nitrogen,  or 
about  .0025%,  which  is  in  the  neighborhood  of  one  one- 
hundredth  the  nitrogen  content  of  a  good  loam. 

A  little  nitrogen  must  inevitably  be  carried  over  in  the 
process  of  dilution,  etc. 

The  plates  were  counted  after  five  days  incubation  at 
room  temperature,  which  prevailed  at  about  33°C. 

The  figures  in  the  adjoining  table  represent  the  count  per 
gram  of  soil  dried  at  100-1 10°C,  to  constant  weight. 

NUMBER  OF  BACTERIA  PER  GRAM  OF  SOIL  DRIED 

TO  CONSTANT  WEIGHT  AT 

100-110°C 

TABLE  I 


Soil  No. 

Nutrient  agar 

Ashby's  medium 

Moisture  Per  cent 

1 

693,000 

1,155,000 

13.44 

2  

94,900 

940,000 

5.23 

3  

3,301,000 

3,056,000 

18.22 

4 

1,673,000 

3,705,000 

16.33 

5  

1,793,000 

2,577,000 

10.77 

6 

210,000 

1,053,000 

5.09 

7  

288,000 

2,138,000 

6.41 

8     

381,000 

448,000 

10.83 

9.. 

1,353,000 

1,466.000 

11.37 

TABLE  I — Continued 


Soil  No. 

Nutrient  agar 

Ashby's  medium 

Moisture  Per  cent 

10 

2,407,000 

150,000 

16.91 

11  

224,000 

693,000 

6.27 

12        .... 

153,000 

54,800 

8.91 

13 

15,300 

404,000 

13.50 

14   

75,800 

162,000 

7.73 

15        

289,000 

133,000 

13.55 

16 

34,000 

10,600 

5.95 

17  

42,900 

219,000 

44.45 

18       

790,000 

11,900 

16.56 

19 

50,600 

562,000 

11.09 

20  

115,000 

399,000 

4.98 

21       

1,530,000 

3,178,000 

15.05 

22 

737,000 

1,989,000 

9.15 

23  

474,000 

830,000 

15.68 

24      

946,000 

2,601,000 

15.44 

25 

165,000 

1,650,000 

9.54 

26  

82,000 

671,000 

15.11 

27 

65,000 

412,000 

7.96 

28  

293,000 

1,084,000 

11.44 

29  

215,000 

420,000 

7.15 

30 

135,000 

135,200 

9.80 

31 

59,000 

462,000 

15.59 

32  

40,700 

184,000 

14.23 

33  

639,000 

575,000 

6.15 

34 

713,000 

594,000 

7.56 

35  
36  

409,000 
445,000 

301,000 
434,000 

7.19 
7.95 

37  .. 

574,000 

1,768,000 

9.55 

38  

1,373,000 

2,146,000 

6.84 

39  

784,000 

753,000 

5.44 

40.  .. 

389,000 

811,000 

5.11 

41 

716,000 

791,000 

6.49 

42  

236,000 

381,000 

6.97 

43  

1,795,000 

2,218,000 

5.32 

44. 

731,000 

1,801,000 

11.19 

45 

637,000 

1,880,000 

428 

46  

416,000 

420,000 

3.89 

47... 

873,000 

484,000 

7.15 

48 

1,886,000 

551,000 

31.08 

49  

1,965,000 

573,000 

38.95 

50  . 

335,000 

1,300,000 

7.71 

51.. 

364,000 

1,287,000 

6.79 

52.. 

74.000 

391.000 

5.43 

8 


TABLE  I — Concluded 


Soil  No. 

Nutrient  Agar 

Ashby's  Medium 

Moisture  Per  cent 

53  

254,000 

637,000 

5.91 

54 

255,000 

210,000 

9  79 

55  

1,602,000 

554,000 

16.98 

56 

1,006,000 

524,000 

8  56 

57  

11,000 

4,500 

9.70 

58                    .    . 

818,000 

549,000 

7.16 

59  

506,000 

60,000 

5.21 

60  

56,500 

9,200 

9.88 

61 

4,224  000 

833  000 

1241 

62  

10 

000 

4.48 

63 

468,000 

60,300 

3  90 

64  

947,000 

904,000 

7.12 

65 

158,000 

70,000 

4.30 

66  

134,000 

17,000 

11.98 

67 

40,000 

30,700 

.84 

68     .               .      . 

10,008,000 

512,000 

16.07 

69  

380,000 

83,600 

7.93 

70.. 

1,850,000 

103,000 

8.13 

In  forty  of  the  samples  the  number  of  bacteria  which 
developed  visible  colonies  on  the  non-proteid  medium  were  in 
excess,  and  in  many  instances  in  great  excess,  of  those  on  the 
nutrient  agar. 

It  will  be  observed  that  in  the  remaining  thirty  samples 
the  number  of  colonies  in  the  Ashby  medium  very  closely 
approximate  those  on  the  nutrient  agar,  and  in  but  few  in- 
stances were  the  colonies  on  nutrient  agar  in  great  preponder- 
ance. 

As  to  what  is  to  be  inferred  from  this  it  is  difficult  to  con- 
jecture; nor  can  we  conclude  that  we  have  here  two  distinct 
flora. 

One  striking  feature  which  invites  attention  to  the  Ashby 
plate,  is  the  variety  of  pigments  observable.  Especially 
characteristic  are  the  blue,  violet,  pink,  purple,  red  and  brown 
colors  which  develop  after  several  days. 

Cladothrix  dichotoma,  which  will  be  considered  later,  is 
one  of  the  most  common  soil  organisms  which  thrive  on  a  lim- 
ited nitrogen  supply.  It  is  a  matter  of  common  knowledge 
that  organisms  growing  under  adverse  conditions,  or  rather  in 


9 

\ 

an  environment  other  than  the  optimum,  lose  at  least  some 
of  their  distinguishing  characteristics.  Many  of  these  organ- 
isms, when  transferred  to  nutrient  agar  slants,  grow  vigorously 
without  pigment  production. 

The  azotobacter  develop  on  the  Ashby  medium  in  great 
profusion,  however,  the  differentiation  of  the  nitrogen-fixing 
bacteria  is  not  sufficiently  established  to  render  an  enumera- 
tion of  them  possible. 

THE  FIXATION  OF  FREE  NITROGEN  BY  BACTERIA 

The  relation  of  bacteria  to  nitrogen  is  perhaps  the  most 
important  problem  which  presents  itself  to  the  agriculturist; 
the  reason  being  that  while  the  nitrogen  forms  a  very  large 
proportion  of  the  constituents  necessary  to  the  building  up  of 
plant  tissue,  it  is  present  in  the  soil  in  a  very  limited  quantity, 
and  consequently  constant  cropping  would  tend  toward  ex- 
hausting the  supply. 

The  fixation  of  free  nitrogen  by  bacteria  is  consummated  in 
two  widely  different  ways,  commonly  designated  as  the  sym- 
biotic and  non-symbiotic  relation.  Symbiosis  involves  a  favor- 
able influence  of  one  species  upon  another.  Many  observers 
contend  that  this  symbiotic  relation  is  detrimental  to  the  host. 
The  symbiotic  relation  existing  between  the  leguminoceae  and 
certain  bacteria  enables  the  former  to  absorb  free  nitrogen 
from  the  air  and  elaborate  it  into  nitrogenous  compounds. 
This  metamorphosis  takes  place  within  the  leguminous  nodules, 
the  earliest  description  of  which  was  given  by  Malpighi  in 
1687,  and  this  observer  referred  to  them  as  galls,  i.  e.,  diseased 
excresences,  an  opinion  also  shared  by  later  writers. 

Treviranus,  in  1853,  was  the  first  to  regard  these  nodules 
as  normal  growths,  and  thirteen  years  later  they  were  studied 
by  Woronin,  who  made  the  subsequently  important  observa- 
tion, that  the  formation  contains  entirely  closed  cells  filled  with 
bacteria. 

Beyerinck,  in  1888,  indubitably  established  the  fungoid 
nature  of  these  bacteria  by  isolating  them  from  the  nodules, 
and  cultivating  them  further  in  artificial  media.  Some  ex- 
hibited certain  slight  but  undeniable  differences  which  were 
not  so  extensive  as  to  make  their  discoverer  feel  justified  in 


10 

classifying  the  organisms  as  separate  species.  Beyerinck 
proposed  the  name  Bacillus  radicicola,  for  these  nodule  pro- 
ducing bacteria;  as  to  whether  there  are  more  than  one  species, 
authorities  are  still  undetermined.  Hereby  is  evolved  a 
rational  system  for  the  continuous  addition  of  nitrogen  to  the 
soil,  an  increase  which  can  not  only  be  enjoyed  and  ap- 
propriated by  the  leguminous  plants,  but  likewise  by  succeed- 
ing vegetable  growth. 

The  non-symbiotic  fixation  of  nitrogen  possesses  the  im- 
portant feature  of  having  more  universal  application.  The 
following  aerobic  species  are  the  most  vigorous  nitrogen  fixing 
organisms  hitherto  discovered:  A.  agilis,  A.  chroococcum, 
A.  vinelandi,  A.  beyerincki,  A.  vitreum,  and  A.  woodstowni. 
Of  these  A.  chroococcum  is  in  all  probability  the  most  common 
in  our  soil.  I  have  isolated  this  organism  from  many  parts  of 
the  state. 

STUDIES  ON  IMPURE  CULTURES 

In  order  to  determine  the  relative  nitrogen-fixing  power  of 
our  soils,  the  aforementioned  samples  were  inoculated  into  Ash- 
by's  medium,  and  the  folio  wing  process  and  technique  followed: 
One  hundred  cubic  centimeters  of  Ashby's  medium  was  meas- 
ured into  250  ccErlenmeyer  flasks,  and  sterilized:  these  flasks, 
therefore,  each  contained  two  grams  of  mannite;  the  medium 
occupying  about  three-fourths  of  an  inch  in  depth  in  the 
bottom  of  the  flask,  there  remained  above  the  surface  an 
abundant  air  space.  These  flasks  were  each  inoculated  with 
one  gram  of  soil,  and  incubated  at  room  temperature,  which 
prevailed  at  about  33°C,  for  twenty-one  days.  At  the  end  of 
this  period  the  entire  contents  of  these  flasks  were  transferred 
to  the  Kjeldahl  apparatus  and  the  total  nitrogen  content 
determined  by  the  Kjeldahl  method,  the  ammonia  being 
distilled  over  into  tenth-normal  sulphuric  acid  and  titrated 
back  with  tenth-normal  sodium  hydroxide,  using  congo  red  as 
indicator.  The  original  nitrogen  content  of  the  soil  was 
determined  by  the  Kjeldahl  method,  using  ten  grams  of 
sample. 

Each  operation  during  the  investigation  was  carefully 
checked  in  order  to  reduce  the  possible  error  to  the  limit  of 
experimental  manipulation.  Samples  number  48  and  49  were 


11 

the  only  typical  muck  soils  available.  It  will  be  observed  that 
these  show  a  fixation  of  4.38  and  5.02  mg  respectively.  One 
mg  of  nitrogen  at  0°C  and  760  mm  pressure  represents  ap- 
proximately 0.80  cubic  centimeters.  The  per  cent  of  nitrogen 
determined  for  these  soils  dried  to  constant  weight,  at  100- 
110°C,  were  .4331  and  .5481,  which  is  very  greatly  in  excess  of 
any  of  the  remaining  soils.  The  average  of  sixteen  soils  of 
which  the  nitrogen  content  ranged  uniformly  above  .2024  per 
cent,  with  a  limit  of  .5481  was  4.91  mg,  while  the  average  of 
sixteen  soils  which  fixed  more  than  4.91  mg  and  which  had  a 
nitrogen  content  of  uniformly  less  than  .2024  per  cent,  was 
6.72  mg.  Soil  number  1  which  had  a  nitrogen  content  of 
.0926  per  cent,  fixed  10.74  mg  which  was  the  highest  value  of 
any.  It  is  reasonable,  therefore,  to  conclude  that  a  soil  which 
contains  much  above  .1000  per  cent  of  nitrogen,  other  things 
being  favorable,  may  equal  or  surpass  any  other  soil  in  nitrogen 
fixing  possibilities.  Probably  the  number  of  azotobacter 
present  in  the  soil  determined  the  speed  of  the  reaction. 

Azotobacter  chroococcum  was  found  to  be  universally 
distributed  over  the  state.  Many  of  the  cultures  which  evinced 
strong  nitrogen-fixing  properties  were  covered  with  an  imperfect 
floating  membrane  of  brownish  color  shading  off  to  almost 
black. 

A.  chroococcum  was  definitely  isolated  from  an  alfalfa 
field  at  a  depth  of  three  feet,  to  which  particular  reference  will 
be  made  later. 

Fungus  growth  developed  on  the  surface  of  the  medium  in 
some  instances.  It  is  significant  that  in  those  overrun  with 
molds  and  similar  vegetation,  the  liquid  frequently  exhibited 
decided  colors,  usually  yellow,  though  in  one  instance  pink. 

The  following  table  shows  the  amount  of  nitrogen  fixed  in 
milligrams  and  the  percentage  of  moisture  and  nitrogen  in 
each  of  the  seventy  samples  of  soil  investigated: 


12 


FIXATION  OF  NITROGEN 
ASHBY'S  MEDIUM 

MANNITE 
TABLE  II 


Soil 
No. 


Moisture 
Per  cent 


N  Fixed 
in  Mg 


Per  cent 
N  in  Sample 


1 13.44 

2 5.23 

3 18.22 

4 16.33 

5 10.77 

6 5.09 

7 6.41 

8 10.83 

9 11.37 

10 16.91 

11 6.27 

12 8.91 

13 13.50 

14 7.73 

15 13.55 

16 5.95 

17 4.45 

18 16.56 

19 11.09 

20 4.98 

21 15.05 

22 9.15 

23 15.69 

24 15.44 

25 9.54 

26 15.11 

27 7.96 

28 11.44 

29 7.15 

30 9.80 

31 15.59 

32 14.23 

33 6.15 

34 7.56 

35 7.18 

36 7.95 

37..  9.55 


10.74 
3.70 
6.05 
4.98 
3.37 
3.04 
9.76 
7.22 
6.94 
5.60 
4.50 
4.97 
0.12 
4.17 
4.08 
0.19 
2.58 
5.57 
5.19 
2.70 
6.32 
5.50 
4.56 
7.30 
4.56 
3.66 
0.59 
4.59 
4.63 
0.61 
0.63 
0.29 
2.83 
6.18 
4.16 
3.95 
5.14 


.0926 
.1981 
.2507 
.1724 
.1104 
.1093 
.1609 
.1987 
.1807 
.2067 
.1537 
.1528 
.1487 
.1493 
.2335 
.1503 
.1418 
.1549 
.1441 
.1431 
.2176 
.2092 
.2507 
.2351 
.2254 
.2024 
.2110 
.2063 
.2156 
.1731 
.1725 
.1686 
.1241 
.2590 
.1462 
.1730 
.1949 


13 

TABLE  II — Continued 


Soil 
No. 


Moisture 
Per  cent 


N  Fixed 
in  Mg 


Per  cent 
N  in  Sample 


38 6.84 

39 5.44 

40 5.11 

41 6.49 

42 6.97 

43 5.32 

44 11.19 

45 4.28 

46 3.89 

47 7.15 

48 31.08 

49 38.95 

50 7.71 

51 6.79 

52 5.43 

53 5.81 

54 9.79 

55 16.98 

56 8.56 

57 9.70 

58 7.16 

59 5.21 

60 '. ...  9.88 

61 12.41 

62 4.48 

63 3.90 

64 7.12 

65 4.30 

66 11.98 

67 .84 

68 16.07 

69 7.93 

70..  8.13 


4.26 
2.74 
5.77 
5.39 
2.21 
8.57 
4.23 
7.72 
7.66 
3.41 
4.38 
5.02 
4.33 
4.93 
0.15 
2.63 
0.29 
5.65 
4.07 
0.00 
4.06 
0.13 
0.00 
6.84 
0.00 
1.36 
0.00 
0.57 
0.00 
0.24 
3.87 
2.73 
5.17 


.1683 
.1720 
.1667 
.1852 
.1477 
.1745 
.1800 
.1153 
.1028 
.1274 
.4331 
.5481 
.1574 
.1876 
.1381 
.1371 
.0472 
.2031 
.1016 
.0172 
.0722 
.0875 
.0176 
.1831 
.0051 
.0425 
.0911 
.1006 
.0347 
.0308 
.1604 
.0598 
.0892 


14 

THE  AVAILABILITY  OF  VARIOUS  COMPOUNDS 
EFFECTING  NITROGEN  FIXATION 

It  having  been  previously  established  that  carbohydrates 
were  essential  for  the  maximum  efficiency  of  nitrogen  fixation, 
many  sugars  have  been  studied  in  these  investigations.  In 
this  connection  I  have  employed  the  following:  mannite, 
maltose,  lactose,  saccharose,  dextrose,  galactose,  levulose, 
arabinose,  dulcite,  sorbit,  raffinose,  rhamnose,  mannose,  ery- 
thrite,  xylose,  quercit,  glycerine,  dextrin,  inulin,  calcium 
lactate,  and  calcium  butyrate.  These  compounds  were  the 
best  obtainable,  mostly  Kahlbaum's  product,  and  were  ac- 
curately assayed  for  nitrogen. 

Ten  soils  which  showed  good  fixation  on  mannite  were 
selected  for  this  purpose:  numbers  1,  2,  7,  10,  24,  34,  41,  43, 
47  and  61.  These  soils  were  inoculated  into  Ashby's  medium 
under  conditions  similar  to  those  followed  in  the  previous 
experiment,  with  the  one  exception  that  the  mannite  was 
replaced  by  a  special  sugar  or  other  compound.  While  it  would 
have  been  highly  desirable  to  have  the  data  for  all  the  sugars 
on  the  ten  samples,  the  prohibitive  price  on  many  rendered 
this  quite  impossible.  An  inspection  of  the  table  shows  the 
ten  highest  averages  as  follows: 

Sorbit 8.32  mg  Dulcite 6.21  mg 

Mannite 7.17  mg  Arabinose 6.14  mg 

Maltose 6.34  mg  Dextrose 5.32  mg 

Mannose 6.32  mg  Galactose 5.08  mg 

Levulose 6.28  mg  Rhamnose 4.92  mg 

The  position  held  by  sorbit  is  probably  only  possible 
because  of  the  remarkable  soil  number  7.  Of  the  disaccharides, 
maltose  gave  the  best  results,  lactose  second,  and  saccharose 
third. 

An  impure  sample  of  maltose  which  we  had  in  the  labora- 
tory, and  which  contained  15  mg  of  nitrogen  per  two  grams  of 
sugar,  fixed  an  average  on  soils  2,  10,  and  41  of  1.02  mg,  while 
the  same  soils  on  pure  maltose  corrected  for  a  very  small  per- 
centage of  nitrogen,  fixed  an  average  of  4.97  mg.  This  may  be 
accepted  as  additional  testimony  that  the  presence  of  nitro- 
genous compounds  in  considerable  amounts  is  not  conducive 
to  high  fixation.  Mannose,  the  aldehyde  of  the  alcohol  man- 


15 

nite,  might  be  expected  to  approach  the  latter  in  fixation,  but 
this  did  not  prove  to  be  the  case,  yet  it  differed  from  maltose 
only  in  the  second  place  of  decimals.  Erythrite  fixed  an 
average,  on  soils  1  and  43  in  twenty-one  days,  of  0.18  mg, 
while  on  soils  10,  24,  34  and  41,  in  fifty-four  days,  an  average 
of  5.59  mg  was  fixed.  The  slow  fermentation  of  this  sugar 
renders  it  useless  for  laboratory  purposes.  Dextrin  and 
inulin  gave  comparable  results  which  were  inconsiderable. 
Probably  twenty-one  days  is  insufficient  to  develop  the  maxi- 
mum efficiency  of  these  polysaccharides. 

Glycerine  in  soils  1  and  43  fixed  an  average  of  1.13  mg  in 
twenty-one  days.  Soils  7,  10,  24,  34,  41,  47,  and  61,  fixed  an 
average  of  6.64  mg  in  thirty-nine  days.  Soil  number  1  fixed 
3. 58  mg  in  thirty-nine  days  and  soil  43  fixed  4. 74  mg  in  the  same 
time,  a  gain  in  the  first  instance  of  2.61  mg  and  in  the  second 
of  2.45  mg  in  eighteen  days.  The  slow  fermentation  of  gly- 
cerine relegates  it  to  the  class  with  erythrite. 

In  the  work  on  mannite  solutions  one  is  struck  with  the 
great  variety  of  odors,  but  perhaps  the  most  characteristic  is 
that  of  butyric  acid.  This  led  me  to  conclude  that  butyric 
acid  or  oxybutric  was  either  one  of  the  splitting  products  of 
mannite,  or  that  according  to  an  early  discovery,  two  mole- 
cules of  lactic  acid  were  changed  to  one  of  butyric  acid,  giving 
off  two  molecules  of  carbon  dioxide  and  two  molecules  of  hy- 
drogen. After  adding  calcium  butyrate  to  Ashby's  medium 
it  was  inoculated  with  soils  1,  10  and  43,  these  yielded  an  aver- 
age fixation  of  0.15  mg.  The  butyrate  therefore  seemed  not 
available  for  carbon  supply.  In  place  of  calcium  butyrate, 
calcium  lactate  was  next  introduced  using  three  grams  to  the 
flask,  an  equivalent  of  2.45  grams  calculated  as  free  lactic  acid. 
The  average  fixation  for  ten  soils  was  3.01  mg.  The  figures 
on  this  compound  do  not  show  the  uniformity  of  the  others, 
although  soils  2,  7,  47  and  61  did  remarkably  well.  The  odor 
of  butyric  acid  was  not  so  pronounced  as  had  been  expected, 
but  some  cultures  showed  unmistakable  evidence  of  its  presence. 

In  the  fermentation  of  mannite  considerable  ethyl  alcohol 
is  split  off.  An  analysis  of  the  total  acidity  revealed  approx- 
imately 30%  acetic  acid  and  70%  butyric  acid. 

The  following  table  shows  the  amount  of  nitrogen  fixed 
when  grown  in  a  medium  containing  the  compounds  listed: 


16 


THE  AVAILABILITY  OF  VARIOUS  COMPOUNDS  FOR 
NITROGEN  FIXATION 

TABLE  III 


" 

Soils 

1 

2 

7 

10 

24 

34 

41 

43 

47 

61 

Maltose.  .  .  . 

5.24 

4.06 

11.21 

4.92 

4.53 

5.70 

5.93 

4.40 

5.79 

11.61 

Lactose.  .  .  . 

3.68 

2.48 

5.36 

4.47 

4.94 

4.21 

2.24 

7.60 

7.62 

Saccharose.  . 

3.98 

3.57 

3.44 

5.48 

3.75 

3.26 

3.45 

2.98 

3.65 

5.79 

Mannite.  .  .  . 

10.74 

3.70 

9.76 

5.60 

7.30 

6.18 

5.39 

8.57 

7.66 

6.84 

Mannose.  .  . 

11.10 

3.50 

10.90 

4.57 

3.35 

5.94 

4.91 

5.58 

10.31 

3.01 

Dextrose.  .  . 

4.85 

3.62 

5.86 

4.75 

3.36 

4.77 

4.41 

4.32 

12.02 

Levulose  

4.84 

4.55 

9.82 

6.62 

5.00 

4.80 

6.94 

5.98 

6.64 

7.69 

Galactose.  .  . 

5.87 

3.17 



4.78 

4.42 

5.35 

4.36 

3.74 

7.48 

6.56 

Raffinose  .  .  . 

5.57 

3.39 

5.71 

4.47 

4.03 

6.44 

5.54 

4.45 



4.53 

Rhamnose.  . 

5.06 

4.66 



3.94 

5.60 

5.61 

4.43 



5.15 

Arabinose.  .  . 

6.41 

6.80 

5.52 

5.02 

5.76 

7.30 

6.23 





Dulcite  

3.28 

14.35 

5.55 

4.69 

5.34 

5.32 

5.00 



Erythrite  .  .  . 

0.23 

7.83 

4.02 

8.07 

2.45 

0.13 





Dextrin 

3.70 

1.88 

3.52 

3.64 

2.02 

Inulin  

3.68 

2.28 

2.02 

2.86 

4.05 



Sorbite  

8.13 

13.27 





3.57 

Xylose  





6.93 

3.84 

3.42 



Quercit  

3.29 

Glycerine  .  .  . 

3.58 



3.37 

6.50 

6.90 

6.53 

7.90 

4.74 

6.14 

9.18 

Calcium 

Lactate  

1.24 

3.39 

3.90 

2.13 

0.88 

2.97 

0.93 

2.55 

4.53 

7.59 

STUDIES  ON  AMMONIFICATION  IN  MIXED 
CULTURE 

The  original  70  samples  were  used  in  connection  with  this 
experiment.  The  medium  consisted  of  a  solution  of  ten  grams 
of  Witte's  peptone  per  liter  of  distilled  water.  One  hundred 
cubic  centimeters  of  this  solution  were  measured  into  flasks 
of  500  cc  capacity,  sterilized,  and  inoculated  with  one  gram  of 
soil.  After  seven  days  incubation  at  33° C,  the  contents  of 
these  Erlenmeyer  flasks  were  transferred  to  the  Kjeldahl 
apparatus,  ten  grams  of  magnesium  oxide  added,  and  the  am- 
monia distilled  over  into  semi-normal  hydrochloric  acid,  and 
titrated  back  with  semi-normal  ammonium  hydroxide,  using 
congo  red  as  indicator.  In  order  to  ascertain  the  percentage 
of  nitrogen  in  the  peptone,  a  composite  sample  was  taken 
from  the  thirteen  stock  bottles,  intimately  mixed,  and  run  by 


17 


the  Kjeldahl  method  in  triplicate.  This  composite  sample 
which  assayed  15.567%  nitrogen  was  used  in  all  ammonifica- 
tion  experiments.  Each  flask  therefore  contained  155.67  mg 
of  nitrogen.  It  will  be  observed  that  in  soils  number  49  and 
61,  over  80%  of  the  nitrogen  was  evolved  as  ammonia.  The 
muck  soil  49  being  a  little  below  the  loam.  A  survey  of  the 
table  indicates  that  those  soils  which  were  especially  active 
in  fixing  nitrogen  also  converted  into  ammonia  more  than 
70%  of  the  available  nitrogen. 

AMMONIFICATION  *  IMPURE  CULTURES 
TABLE  IV 


Soil 
No. 

Nitrogen  Evolved 
as  Ammonia 
inMg 

Per  cent  of 
Nitrogen  Evolved 
as  Ammonia 

1       .    

11726 

75  32 

2 

105  77 

67  <U 

3  

122  80 

78  88 

4 

116  00 

74  51 

5  

120.13 

77  16 

6  

115.03 

73  88 

7 

11047 

70  Qfi 

8  

120.34 

77  30 

9  

121  75 

78  21 

10     . 

118  87 

76  26 

11  

109.77 

70  51 

12  

96  11 

61  73 

13  

106  67 

68  53 

14. 

96  67 

62  09 

15  

111.66 

71  72 

16  

106  19 

68  21 

17. 

112  30 

72  13 

18  

113.13 

72  67 

19  

106  61 

68  48 

20  

86  02 

55  25 

21 

108  65 

fiq  70 

22  

108  51 

69  70 

23  

109  00 

70  01 

24  

109.00 

70  01 

25  

102  97 

66  14 

26  

103  25 

66  32 

27  

102.62 

6592 

28.. 

102.97 

66.14 

18 


TABLE  IV— Continued 


Soil 
No. 

Nitrogen  Evolved 
as  Ammonia 
in  Mg 

Per  cent  of 
Nitrogen  Evolved 
as  Ammonia 

29  

9345 

60  03 

30  

93.80 

6025 

31  

115  58 

74  24 

32  

110.12     . 

70.74 

33  

118.66 

7622 

34  

120.55 

77.50 

35  

121.19 

77  85 

36  

117.68 

75.59 

37  

117.26 

7532 

38  

112.22 

72.08 

39  

113.48 

73  89 

40  

102.76 

66.01 

41. 

122.10 

7843 

42  

118.94 

76.40 

43. 

110.75 

71  14 

44  

120.06 

77.12 

45 

110.33 

70.87 

46  

107.81 

69.25 

47  

110.54 

71.00 

48  

113.84 

73.12 

49  

126.09 

80.99 

50  

99.68 

64.03 

51  

118.59 

76.18 

52.    .                                  

111.59 

71.68 

53  

100.95 

64.84 

54 

84.76 

54.44 

55  

98.84 

63.49 

56  

77.75 

49.93 

57  

82.03 

52.69 

58  

92.89 

59.66 

59  

101.29 

65.06 

60  

34.60 

22.22 

61  

126.44 

81.22 

62  

37.40 

24.02 

63  

71.58 

45.98 

64  

92.03 

59.75 

65  

79.58 

51.11 

66  

27.04 

17.36 

67  

99.54 

63.94 

68  

105.63 

67.85 

69  

94.64 

60.79 

70.. 

95.90 

61.60 

19 


THE  AMMONIFICATION  OF  MEAT-EXTRACT 

As  a  comparison  between  the  availability  of  meat-extract 
and  peptone  nitrogen  for  ammonification  experiments,  a 
medium  consisting  of  19.85  grams  Liebig's  extract  of  meat 
per  liter  of  distilled  water,  was  used.  The  meat-extract  as- 
sayed 7.94%  nitrogen,  which  was  approximately  the  peptone- 
nitrogen  content. 


TABLE     SHOWING     COMPARATIVE     WEIGHTS 
NITROGEN  EVOLVED  AS  AMMONIA  IN  PEP- 
TONE    AND     MEAT-EXTRACT     MEDIA 
HAVING  SAME  NITROGEN  CONTENT 

TABLE  V 


OF 


Soil 
No. 

PEPTONE 

MEAT-EXTRACT 

Per  cent 
Difference 

Mg 
N  Evolved 

Per  cent 
N  Evolved 

Mg 
N  Evolved 

Per  cent 
N  Evolved 

9 

104.23 
107.03 
92.74 
107.73 
120.63 

66.10 
67.87 
58.81 
68.32 
76.50 

121.75 
117.26 
110.75 
126.09 
126.44 

78.21 
75.32 
71.14 
80.99 
81.22 

12.11 
7.45 
12.33 
12.67 

4.72 

37 

43  

49 

61.  . 

The  ammonification  in  the  meat-extract  medium  appears 
to  be  uniformly  lower  than  in  the  peptone  solution,  and  the 
difference  in  per  cent  in  soils  9,  43  and  49  is  constant.  An 
attempt  was  made  to  use  lecithin  as  a  culture  medium  for  sim- 
ilar experiments,  but  the  nitrogen  content  being  low  (1.80)%, 
and  the  insolubility  so  great,  the  attempt  was  finally  aban- 
doned. 


THE  DEVIATION  OF  NASCENT  HYDROGEN   FROM 

THE  PROTEIN  NITROGEN  EFFECTED  BY  THE 

PRESENCE  OF  NITRATES  AND   NITRITES 

Fifteen  flasks  each  containing  100  cc  of  the  1%  peptone 
solution,  were  inoculated  with  one  gram  of  soil  as  indicated: 
To  each  of  the  second  set  of  five,  was  added  one  gram  of  potas- 
sium nitrate.  Similarly  to  the  third  set  of  five,  was  added 


20 


one  gram  of  potassium  nitrite.  These  flasks  were  incubated 
at  33°C  for  seven  days,  the  contents  then  transferred  to  the 
Kjeldahl  apparatus  and  the  ammonia  determined  as  in  the  pre- 
vious work. 

TABLE  VI 


S 

OIL  No. 

9 

37 

43 

49 

61 

Peptone 

72.40 

65.92 

72.76 

76.49 

74.74 

Peptone  plus  pot.  nitrate  
Peptone  plus  pot.  nitrite  

39.86 
31.18 

32.80 
25.96 

40.90 
33.97 

36.26 
22.67 

43.87 
30.91 

The  figures  in  the  above  table  represent  the  percentage  of 
nitrogen  evolved  as  ammonia.  The  conclusion  is  inevitable 
that  considerable  quantities  of  nitrates  or  nitrites  cannot  exist 
in  the  soil  together  with  a  high  percentage  of  protein  nitrogen. 
The  ammonification  equilibrium  is  likewise  disturbed  by  an 
excess  of  nitrates  or  nitrites. 

The  figures  in  the  above  table  seem  to  indicate  that  the 
hydrogen  together  with  a  large  proportion  of  the  nitrogen, 
perhaps  partly  because  of  the  violence  of  the  reaction,  on  being 
set  free  escapes  as  free  hydrogen  and  nitrogen.  We  have  in 
each  column  of  the  table  a  consistently  decreasing  percentage 
of  ammonification.  The  relation  between  the  peptone  and  pep- 
tone-nitrate being  uniformly  greater  than  the  relation  between 
the  peptone-nitrate  and  peptone-nitrite.  This  uniformity  is 
pronounced. 

STUDIES  ON  THE  REDUCTION  OF  NITRATES 

IMPURE  CULTURES:  One  hundred  cubic  centimeters  of 
Ashby's  medium  without  the  carbohydrate,  was  measured 
into  Erlenmeyer  flasks  of  250  cc  capacity.  To  each  flask  was 
added  varying  amounts  of  mannite,  dextrose  and  potassium 
salts  as  indicated  in  the  table.  These  flasks  were  inoculated 
with  different  soils  and  kept  at  28°C. 


2 

.1 

> 

be 
1 


•s 


21 


TABLE  VII 


Soil 
No. 

Mannite 

KNO3 

Day  on  which 
Nitrate  was  Found 
to  be  Reduced 

5  

2  grams 

1  gram 

5th 

10          

2  grams 

1  gram 

5th 

19 

2  grams 

1  gram 

5th 

22  

2  grams 

1  gram 

5th 

37 

2  grams 

1  gram 

5th 

61  

2  grams 

1  gram 

5th 

5  

4  grams 

1  gram 

4th 

10 

6  grams 

200  mg 

4th 

19  

4  grams 

200  mg 

4th 

22  

4  grams 

100  mg 

4th 

31 

4  grams 

500  mg 

4th 

37  

6  grams 

200  mg 

4th 

61.  . 

6  grams 

200  mg 

4th 

61 

4  grams 

250  mg 

4th 

Dextrose 

37  

2  grams 

1  gram 

5th 

49 

2  grams 

1  gram 

5th 

61  

2  grams 

1  gram 

5th 

The  development  within  this  medium  was  especially  rapid, 
the  evolution  of  gas  beginning  after  the  second  day  and  continu- 
ing with  increased  vigor  for  some  time.  In  the  flasks  which 
contained  the  gi  eater  amounts  of  carbohydrate  the  evidence 
of  powerful  reduction  was  most  pronounced,  the  surface  being 
rapidly  overspread  with  fusarium  and  other  fungi,  which  were 
not  apparent  on  those  with  lower  sugar  content.  The  evolu- 
tion of  gas  was  so  violent  in  some  cases  as  to  force  the  felt- 
like  growth  from  the  surface,  high  above  the  liquid,  as  il- 
lustrated in  the  accompanying  figure.  The  evidence  here 
presented  indicates  that  the  reduction  of  nitrates  is  carried  on 
with  great  vigor  in  the  presence  of  considerable  quantities  of 
carbonaceous  material.  No  appreciable  difference  could  be 
detected  in  favor  of  either  mannite  or  dextrose. 


22 


REDUCTION  OF  NITRATES  CONTINUED 

IMPURE  CULTURES:  The  medium  used  in  this  experiment 
was  the  1%  peptone  solution  to  which  was  added  one  gram  of 
potassium  nitrate  per  liter.  This  solution  was  distributed  in 
150  cc.  Erlenmeyer  flasks  in  amounts  of  50  cc  each,  and  after 
inoculation  with  one  gram  of  soil  was  incubated  at  33°C  for 
the  length  of  time  and  with  the  results  recorded  below  in  table 
No.  VIII. 

TABLE  VIII 


Soil 
No. 

Time 

Per  cent 
of  Nitrite 

1                             

30  hours 

37  50 

2           

30  hours 

44.00 

3                                 

30  hours 

18  75 

4              

30  hours 

31.25 

5  

30  hours 

31.25 

6                  

30  hours 

25.00 

7  

30  hours 

40.00 

8                  

30  hours 

15.00 

9       

30  hours 

31.25 

10                    

24  hours 

50.00 

11  

24  hours 

35.00 

12  

24  hours 

37.50 

13.    .    .    

24  hours 

31.30 

14  

24  hours 

37.50 

15  

24  hours 

50.00 

16                           

24  hours 

37.50 

17            

12  hours 

50.00 

18  

24  hours 

37.50 

19                

24  hours 

43.80 

20                                   

24  hours 

50.00 

21             

24  hours 

21.88 

22       

24  hours 

15.00 

23         

24  hours 

21.88 

24                                     

24  hours 

18.75 

25     

24  hours 

18.75 

26                    

24  hours 

18.75 

27                                       

24  hours 

15.25 

28       

24  hours 

21.88 

29                               

24  hours 

37.50 

30  

24  hours 

15.25 

31              

24  hours 

18.75 

32.. 

24  hours 

18.75 

23 

TABLE  VIII— Continued 


Soil 
No. 

Time 

Per  cent 
of  Nitrite 

33  

30  hours 

1875 

34  

30  hours 

18  75 

35  

30  hours 

22  00 

36  

30  hours 

2500 

37  

30  hours 

22  50 

38  

30  hours 

1875 

39  

30  hours 

18  75 

40  

30  hours 

18.75 

41....  

30  hours 

22  50 

42  

30  hours 

21.87 

43  

30  hours 

1563 

44  

30  hours 

21  87 

45  

36  hours 

43.75 

46  

36  hours 

4375 

47  

36  hours 

31  25 

48  

36  hours 

8.75 

49  .      .. 

36  hours 

33  30 

50  

36  hours 

22  50 

51  

36  hours 

27  50 

52  

36  hours 

33  30 

53  

36  hours 

.45 

54  

36  hours 

4375 

55  

24  hours 

43  75 

56  

24  hours 

46.25 

57  

24  hours 

43  75 

58  

24  hours 

4625 

59  

24  hours 

43.74 

60  

24  hours 

320 

61  

24  hours 

4625 

62  

24  hours 

43.75 

63  

24  hours 

46.25 

64  

24  hours 

43  75 

65  

24  hours 

43.75 

66  

24  hours 

31.25 

67  

24  hours 

43  75 

68  

24  hours 

43.75 

69  

24  hours 

43.75 

70  

24  hours 

43.75 

The  following  solutions  were  used  for  determining  the  am- 
ount of  nitrite  present: 


24 

I     a-Naphthylamine 1.00  gram 

Distilled  water 100.00  grams 

II    Sulphanilic  acid 50  gram 

Dilute  acetic  acid 150.00  cc 

These  solutions  were  kept  in  separate  glass  stoppered 
bottles. 

In  the  performance  of  the  operation  5  cc  of  the  culture 
medium  were  transferred  to  a  Nesslerizing  tube  by  means  of  a 
pipette,  about  25  cc  of  distilled  water  added,  and  1  cc  of  each 
of  the  above  solutions  introduced.  The  solution  was  brought 
up  to  the  50  or  100  cc  mark  with  distilled  water.  The  quan- 
titative estimation  of  the  nitrite  was  determined  by  the  col- 
orimic  method.  Every  sample  of  soil  without  exception  con- 
tained bacteria  which  reduced  nitrates  to  nitrites.  The  odors 
emanating  from  these  cultures  and  from  the  ammonfication 
experiments  were  exceedingly  offensive.  To  determine  the 
possibility  of  reduction  of  nitrates  in  soil  infusion,  100  cc  of 
distilled  water  was  measured  into  each  of  nine  250  cc  Erlen- 
meyer  flasks;  to  each  flask  was  added  100  mg  of  potassium  ni- 
trate free  from  nitrite.  These  flasks  were  inoculated  with 
soils  5,  47,  49,  51,  54,  55,  57,  60  and  61  respectively.  They 
were  incubated  at  33°C  for  seven  days.  Tests  were  then  made 
for  nitrites  and  all  without  exception  were  found  to  be  negative. 
No  reduction  is  therefore  probable  except  in  the  presence  of 
considerable  available  nitrogen. 

STUDIES  ON  THE  REDUCTION  OF  NITRITES 

IMPURE  CULTURES:  One  hundred  cubic  centimeters  of 
Ashby's  medium  without  the  carbohydrate,  were  measured 
into  250  cc  Erlenmeyer  flasks.  To  each  flask  was  added  vary- 
ing amounts  of  mannite,  dextrose  and  potassium  salts  as  in- 
dicated. These  flasks  were  inoculated  with  one  gram  of  the 
different  soils  and  incubated  at  28°C  for  the  length  of  time  and 
with  the  results  recorded  below  in  table  No.  IX. 


25 

TABLE  IX 


Soil 
No. 

Mannite 

KNO2 

Day  on  which 
Nitrite  was  Found 
to  be  Reduced 

10  

2  grams 

1  gram 

11 

19           

2  grams 

1  gram 

11 

22 

2  grams 

1  gram 

11 

61  

2  grams 

1  gram 

11 

Soil 
No. 

Dextrose 

KN02 

37 

2  grams 

1  gram 

15 

49  

2  grams 

1  gram 

15 

61 

2  grams 

1  gram 

15 

61.. 

4  errams 

1  crram 

18 

Soils  number  37,  49,  and  61  in  dextrose  gave  on  the 
eleventh  day  very  appreciable  reaction  for  nitrite;  not  until 
the  fifteenth  day  did  this  totally  disappear.  An  inspection  of 
this  table  conveys  the  idea  at  once  that  the  nitrite  disappears 
from  the  mannite-containing  medium  more  rapidly  than  from 
the  dextrose. 


THE  REDUCTION  OF  NITRITES  IN  PEPTONE 
SOLUTION 

IMPURE  CULTURES:  Fifty  cubic  centimeters  of  a  medium 
containing  ten  grams  of  peptone,  together  with  one  gram  of 
potassium  nitrite  per  liter  of  distilled  water,  were  measured 
into  150  cc  Erlenmeyer  flasks  and  sterilized.  These  flasks 
were  each  inoculated  with  one  gram  of  soil  and  incubated  at 
33°C.  After  periods  of  time  as  indicated  in  the  table  number 
X,  the  solutions  were  tested  for  the  presence  of  nitrites.  On 
sterilizing  peptone-nitrite  solution  in  the  Arnold,  the  liquid 
assumes  a  more  decided  yellow  color  than  the  peptone-nitrate 
solution.  A  quantitative  determination  shows  that  a  portion 
of  the  nitrite  has  combined  with  the  peptone,  therefore  the 
results  are  invariably  low.  All  of  the  soils  contained  organisms 
which  rapidly  reduced  nitrites  to  free  nitrogen.  The  rapidity 


26 


of  this  reaction  is  very  marked.  After  twenty-four  hours 
the  surface  of  the  liquid  is  covered  with  foam,  and  at  the  end 
of  two  days  very  little  nitrite  remains.  Those  cultures  in 
which  the  evolution  of  gas  was  most  pronounced,  evolved 
disagreeable  odors;  while  those  which  developed  but  slight 
activity  were  comparatively  odorless.  But  few  species  of 
bacteria  reduce  nitrites  to  free  nitrogen  in  straight  peptone 
media.  The  fluorescens  group  are  of  special  importance  in 
producing  this  change.  Representatives  of  this  class  were 
isolated  from  many  of  these  soils  and  were  probably  indigenous 
to  all. 

TABLE  X 


Soil 
No. 

Time 

Per  cent 
of  Nitrite 

1  

2  days 

28.00 

2 

2  days 

17.60 

3  ,  

2  days 

0.00 

4  

2  days 

14.00 

5  

2  days 

8.00 

6  

2  days 

10.00 

7    ...     

2  days 

14.00 

8  

2  days 

0.00 

9    .  .      

2  days 

0.00 

10..  

2  days 

12.00 

11                 .         

2  days 

32.00 

12  

2  days 

14.00 

13                            

2  days 

50.00 

14  

2  days 

12.00 

15 

2  days 

56.00 

16  

2  days 

24.00 

17 

2  days 

28.00 

18  

2  days 

4.00 

19 

2  days 

56.00 

20  

2  days 

12.00 

21..  . 

5  days 

2.40 

22  

5  days 

0.00 

23  

5  days 

0.00 

24                              

5  days 

0.00 

25  

5  days 

5.60 

26.  .                       

5  days 

0.00 

27 

5  days 

0.00 

28  

5  days 

0.00 

29.. 

5  days 

0.00 

27 

TABLE  X — Continued 


Soil 
No. 

Time 

Per  cent 
of  Nitrite 

30 

5  days 

0  00 

31 

5  days 

0  00 

32   

5  days 

0  00 

33 

6  days 

0  00 

34 

6  days 

0  00 

35  

6  days 

000 

36 

6  days 

0  00 

37  

6  days 

0.00 

38   

6  days 

ooo 

39 

6  days 

o  oo 

40  

6  days 

0  00 

41  

6  davs 

0  00 

42  

6  days 

0.00 

43  

6  davs 

000 

44     ....... 

6  days 

0  00 

45  

3  days 

0.00 

46  

3  days 

0  00 

47 

6  days 

0  00 

48  

6  days 

0.00 

49  

6  days 

24  00 

50  

6  days 

0.00 

51  

6  davs 

12  00 

52  

6  days 

0.00 

53  

6  days 

0.00 

54  ...   . 

4  days 

0.00 

55  

4  days 

0  00 

56  

4  days 

0.00 

57  

4  days 

44  00 

58  

4  days 

0  00 

59  

4  days 

0.00 

60  

4  days 

50  00 

61  

4  days 

0  00 

62  

4  days 

70.00 

63  

4  davs 

0.00 

64  

4  days 

000 

65  

4  days 

0  00 

66  

4  davs 

63  00 

67  

4  days 

000 

68  

4  days 

0  00 

69  

4  days 

0.00 

70  

4  days 

0.00 

28 
BACTERIA  CONTENT  OF  THE  SUBSOIL 

An  investigation  was  begun  at  the  instigation  of  Hon. 
George  Coupland,  Regent  of  the  University  of  Nebraska,  to 
determine  the  lowest  depth  of  subsoil  in  which  micro-organisms 
might  be  found.  In  order  to  facilitate  the  sampling  it  was 
necessary  that  the  subsoil,  to  the  total  depth  projected,  should 
be  exposed.  Therefore  a  hole,  which  was  approximately 
four  feet  in  diameter  and  twenty-one  feet  deep,  was  dug  in  an 
alfalfa  field  on  the  farm  of  Mr.  Coupland.  Twenty-one 
samples  were  taken  at  intervals  of  one  foot  along  this  per- 
pendicular line.  Four  surface  samples  were  also  taken,  east, 
west,  north  and  south  of  the  excavation,  at  a  distance  of  ten 
feet.  These  samples  represent  two  and  four  inch  depths. 
All  samples  were  plated  both  on  nutrient  agar  and  on  Ashby's 
medium.  Table  number  XI,  shows  no  striking  variation  to 
the  sixth  foot,  except  that  the  fourth  level  is  abnormally  high. 
The  oscillation  thence  to  the  thirteenth  level  is  neither  sur- 
prising nor  unprecedented,  but  the  great  preponderance  on 
the  thirteenth  level  is  unaccountable.  No  visible  stratum  of 
impervious  earth  was  observed.  Alfalfa  roots  penetrated  to 
the  lowest  depth.  While  the  number  of  bacteria  on  the  thir- 
teenth level  was  very  great,  yet,  the  flora  was  little  diversified. 
Cladothrix  dichotoma  being  the  principal  representative. 
Undoubted  azotobacter  were  isolated  from  the  third  level. 
Of  the  different  species  isolated  from  the  above  samples, 
five  fermented  lactose  bouillon  with  gas  production.  Fifty- 
eight  per  cent  reduced  nitrates  to  nitrites. 


Excavation  showing  method  of  sampling. 


29 


NUMBER   OF   BACTERIA   PER   GRAM   OF   SUBSOIL 

DRIED  TO  CONSTANT  WEIGHT  AT 

100— 110°C 

TABLE  XI 


Depth 

Nutrient  Agar 

Ashby's  Medium 

2  inches  . 

2,500.000 

610.000 

4  inches 

660  000 

458,000 

1  foot  

290,000 

417,000 

2  feet                  

282,000 

250,000 

3  feet 

169  000 

185  000 

4  feet  

277,000 

210,000 

5  feet 

156,000 

114,000 

6  feet  

66,000 

47,000 

7  feet           

11,000 

2,000 

8  feet 

7.400 

7,100 

9  feet   

700 

300 

10  feet 

1,200 

1,000 

11  feet 

4700 

2,700 

12  feet       

1,200 

2,600 

13  feet 

26,500 

116,000 

14  feet  

50 

000 

15  feet 

000 

000 

16  feet  

000 

000 

18  feet     

000 

000 

19  feet 

000 

000 

20  feet  

000 

000 

THE  FATE  OF  UREA  IN  THE  SOIL 

IMPURE  CULTURES:  To  asertain  the  changes  which  take 
place  when  urea  is  added  to  nitrogenous  media,  100  cc  of  the 
1%  peptone  solution  were  measured  into  250  cc  Erlenmeyer 
flasks  and  sterilized.  To  each  flask  was  added  one  gram  of 
urea.  They  were  then  inoculated  with  soils  1,  10,  13,  25,  27, 
28,  34,  35,  45,  49,  61  and  68.  After  seven  days  incubation 
at  33°C,  each  flask  gave  off  strong  odor  of  ammonia.  An 
inquiry  into  the  presence  of  carbonate  was  then  instituted 
with  positive  result.  I  therefore  conclude  that  the  organisms 
which  transform  urea  to  ammonium  carbonate  in  the  presence 
of  abundant  available  nitrogen  supply,  are  universally  distri- 
buted within  our  soil.  To  ascertain  the  trend  of  the  reaction 


30 

in  nitrogen-poor  media,  flasks  were  filled  with  Ashby's  medium 
as  before,  one  gram  of  urea  introduced,  and  inoculated 
as  in  the  nitrogenous  medium.  After  forty  eight  hours  the 
surface  was  covered  with  gas  bubbles  and  on  the  fourth  day 
a  strong  odor  of  ammonia  was  evolved  from  each  flask.  It 
therefore  appears  that  in  the  presence  of  an  abundant  nitrogen 
supply  urea  is  converted  into  ammonium  carbonate,  and  that 
this  process  is  not  impeded  by  the  presence  of  carbohydrate 
in  great  excess,  but  is  rather  promoted,  even  though  the 
nitrogen  content  be  very  small. 

Several  flasks  of  Ashby's  medium  were  inoculated  with 
soils  and  thio-urea  introduced  in  place  of  urea.  The  growth 
in  these  flasks  was  much  less  pronounced  than  in  those  con- 
taining urea.  Evidently  ammonium  sulphite  was  not  formed. 

Hippuric  acid  is  split,  in  nitrogenous  media,  into  benzoic 
acid  and  amino-acetic  acid.  Several  flasks  of  Ashby's  medium 
were  inoculated  with  soil  and  one  gram  of  hippuric  acid  in- 
troduced. After  a  few  days  the  surface  was  overgrown  with 
molds,  later  a  vigorous  evolution  of  carbon  dioxide  was  per- 
ceptible, the  overlying  growth  being  forced  high  in  the  flask. 
In  a  second  experiment  the  hippuric  acid  was  neutralized  with 
sodium  hydroxide  before  being  transferred  to  the  Ashby's 
medium.  The  splitting  of  the  hippuric  acid  molecule  into 
benzoic  acid  and  amino-acetic  acid,  and  the  subsequent  union 
of  the  benzoic  acid  and  calcium  carbonate  to  form  calcium 
benzoate,  necessitates  the  liberation  of  considerable  quanti- 
ties of  carbon  dioxide.  Amino-acetic  acid  (Glycocoll)  is 
reduced  by  soil  bacteria  to  ammonia  and  acetic  acid,  this  re- 
duction is  consummated  both  in  nitrogenous  and  non-nitro- 
genous media. 

THE  REDUCTION  OF  NITRATES  TO  NITRITES 

The  breaking  down  of  organic  compounds  by  bacterial 
agency,  falls  under  two  categories;  simple  cleavage,  and  partial 
elementary  disintegration  of  the  proteid  and  carbohydrate 
molecule.  In  the  first  category  we  are  concerned  with  the 
simple  splitting  off  of  groups  from  the  original  relatively  com- 
plex molecule.  Among  the  cleavage  products  may  be  men- 
tioned alcohols,  esters,  mercaptans,  amino-acids,  phenol, 


31 

skatol,  indol,  acids,  glycols,  etc.  The  formation  of  nascent 
hydrogen  by  the  action  of  destructive  organisms  on  car- 
bohydrate and  proteid  compounds  may  be  best  illustrated  by 
a  careful  study  of  the  products  obtained  by  the  destructive 
distillation  of  coal,  wood  and  other  products  of  animal  and 
vegetable  origin.  In  the  destructive  distillation  of  coal  we 
get  as  products:  02,  H2,  N2,  S2,  Cx,  H20,  NH3,  H2S,  CH4,  CO, 
C02,  C2H2,  C2H4,  C6H6,  CS2,  etc.  All  of  these  products  are 
obtained  in  small  or  large  amounts  depending  on  the  com- 
position of  the  coal,  character  of  heating,  etc.  Can  these 
products  be  explained  in  any  other  way  than  that  the  complex 
proteid  molecules  undergo  in  this  process  of  destructive  dis- 
tillation, complete  disintegration  into  their  constitutent 
elements:  C,  0,  H,  N,  S.?  These  elements  must  exist  mo- 
mentarily in  the  active  or  nascent  state.  Because  of  their 
great  chemical  affinity  these  active  elements  then  combine 
with  each  other  to  form  inactive  molecules  which  are  free  to 
pass  off  from  the  sphere  of  action.  In  the  formation  of  these 
simple  molecules  some  of  the  atoms  have  combined  with 
different  atoms,  while  some  have  combined  with  other 
atoms  of  the  same  kind.  As  a  result  of  the  first  method 
we  get:  H20,  NH3,  H2S,  CO,  C02,  C2H4,  CH4,  etc.  As  a 
result  of  the  second  method  we  get:  Cx  (coke  or  soot),  N2,  H2, 
02,  S2,  etc. 

For  the  reduction  of  nitrates  in  pure  culture,  a  medium 
consisting  of  peptone  1%  and  potassium  nitrate  1%  was  em- 
ployed. 10  cc  of  this  medium  were  introduced  into  each 
tube  and  sterilized.  These  tubes  were  inoculated  with  the 
various  organisms  and  incubated  at  33°C  for  ten  days.  At 
the  expiration  of  this  time  they  were  tested  for  the  presence 
of  nitrates  and  nitrites.  The  presence  of  nitrate  was  deter- 
mined by  the  addition  of  a  1%  solution  of  diphenylamine  in 
pure  concentrated  sulphuric  acid.  The  nitrite  was  deter- 
mined according  to  the  method  used  in  the  previous  work 
on  impure  cultures.  Those  cultures  which  failed  to  reduce 
nitrates  to  nitrites  within  ten  days  were  duplicated  and  the 
time  extended  to  thirty  days  for  a  final  reading. 

The  organisms  in  the  following  list  were  obtained  from  the 
celebrated  Krai  collection,  Vienna  Austria,  Prof.  Kraus  Cura- 
tor; and  from  the  American  Museum  of  Natural  History, 


32 

New  York,  Prof.  C.  E.  A.  Winslow  Curator.  No  attempt 
was  made  to  determine  whether  they  were  true  to  name,  the 
only  precaution  being  that  they  were  in  pure  culture.  It  is 
quite  improbable  that  any  considerable  collection  of  species 
would  be  assembled  without  some  repetition  under  different 
names.  Not  all  the  organisms  listed  are  strictly  soil  bacteria, 
several  of  the  intestinal  group  being  purposely  included.  A 
few  have  no  connection  with  soil  fertility  whatever. 

CATALOG  OF  ORGANISMS 

1.  Bacterium  acetosum     [Henneberg] 

2.  Bacterium  lactis  aerogenes     [Escherich] 

3.  Bacillus  brassicae  acidae 

4.  Micrococcus  agilis     [Ali-Cohen] 

5.  Bacillus  acidi  lactici     [Hueppe] 

6.  Micrococcus  albidus 

7.  Bacillus  amylovorus 

8.  Bacillus  anthracis 

9.  Bacillus  pseudo-anthracis 

10.  Bacillus  anthracoides 

11.  Bacterium  annulatum  A 

12.  Bacterium  annulatum  B 

13.  Bacillus  aquatile 

14.  Bacillus  arborescens     [Frankland] 

15.  Bacillus  argentinensis     [Kayser] 

16.  Micrococcus  ascoformans 

17.  Bacillus  asterosporus 

18.  Bacterium  aurantiacus 

19.  Sarcina  aurantiaca 

20.  Bacillus  Baccarinii     [Macchiati] 

21.  Bacterium  beticolum 

22.  Micrococcus  brunneus 

23.  Bacillus  budapestinensis     [Ajtay] 

24.  Bacillus  butyricus     [Hueppe] 

25.  Bacillus  candicans 

26.  Micrococcus  candicans     [Flugge] 

27.  Monila  Candida 

28.  Bacillus  campestris 

29.  Rhodobacillus  capsulatus 


33 


30.  Bacillus  cereus     [Frankland] 

31.  Bacillus  cereulens 

32.  Micrococcus  cereus 

33.  Micrococcus  carneus 

34.  Micrococcus  cinnabareus 

35.  Bacillus  cloacae     [Jordan] 

36.  Micrococcus  citreus 

37.  Bacillus  constrictus 

38.  Micrococcus  concentricus 

39.  Bacillus  coli  commune     [Kruse] 

40.  Bacillus  coli-anaerogenes 

41.  Bacillus  carotovorus     [Jones] 

42.  Bacillus  cyanogenes     [Hueppe] 

43.  Bacillus  cylindrosporus     [Burchard] 

44.  Bacillus  creusus 

45.  Bacillus  cyaneus 

46.  Bacterium  crysogloia 

47.  Bacillus  denitrificans 

48.  Bacillus  dendroides 

49.  Pseudomonas  destructans 

50.  Bacillus  disciformans 

51.  Bacillus  enteritidis     [Gaertner] 

52.  Bacillus  esterigenes     [Krai] 

53.  Bacillus  esterigenes  A 

54.  Bacillus  esterigenes  D 

55.  Bacterium  lactis  erythrogenes     [Grotenfeldt] 

56.  Bacillus  ethacinicus 

57.  Bacillus  ethaceto  succinicus 

58.  Bacillus  ferruginous 

59.  Bacillus  faecalis  alcaligenes     [Petruschky] 

60.  Sarcina  flava 

61.  Micrococcus  flavus     [Flugge] 

62.  Bacillus  flavidus 

63.  Bacterium  filiforme     [Henrici] 

64.  Bacterium  filifaciens     [H.  Jensen] 

65.  Bacillus  Fitzianus 

66.  Bacillus  fluorescens  liquefaciens  [Flugge] 

67.  Bacillus  fluorescens  non  liquefaciens 

68.  Bacillus  fluorescens  tenuis 

69.  Bacillus  Frostii 


34 


70.  Bacillus  fuchsinus    [Balkhout] 

71.  Sarcina  gasformans 

72.  Bacillus  graviolens    [A.  Meyer  et  Gottheil] 

73.  Bacterium  aquatile  griseum 

74.  Micrococcus  grossus    [Henrici] 

75.  Bacterium  Hartlebi    [H.  Jensen] 

76.  Bacillus  havaniensis 

77.  Bacillus  herbicoli  aureus 

78.  Bacillus  helvolus    [Zimmermann] 

79.  Bacillus  hoagii 

80.  Bacillus  hyponitrous 

81.  Bacillus  immobile 

82.  Bacillus  indicus 

83.  Bacillus  indigoferus    [Voges] 

84.  Bacillus  irritans 

85.  Bacillus  ivilans 

86.  Bacillus  jasminocyaneus 

87.  Bacillus  juglandis 

88.  Bacillus  kiliensis 

89.  Bacillus  lactis 

90.  Bacillus  lactorubefaciens    [Gruber] 

91.  Bacillus  lateritia 

92.  Bacillus  levans 

93.  Bacillus  lactis  amari  liquefaciens    [Freudenreich] 

94.  Bacillus  liodermos 

95.  Bacillus  limosus 

96.  Sarcina  liquefaciens    [Frankland] 

97.  Bacillus  liquefaciens 

98.  Bacillus  lactis  niger 

99.  Bacillus  liquefaciens  niger 

100.  Bacillus  loxosus    [Burchard] 

101.  Bacterium  aquatile  gasformans  non  liquefaciens 

102.  Micrococcus  luteus 

103.  Sarcina  lutea 

104.  Streptococcus  luteus  liquefaciens 

105.  Bacillus  maidis 

106.  Bacillus  melonis    [Winslow] 

107.  Bacillus  mesentericus  fuscus 

108.  Bacillus  mesentericus  niger 

109.  Bacillus  mesentericus  ruber 


35 

110.  Bacillus  mesentericus  vulgatus 

111.  Bacillus  megatherium    [De  Bary] 

112.  Bacillus  miniaceus     [Zimmermann] 

113.  Bacillus  proteus  mirabilis 

114.  Sarcina  mobilis 

115.  Moellers  grass  bacillus,  Mist. 

116.  Bacterium  muris     [E.  Klein] 

117.  Bacillus  mycoides     [Flugge] 

118.  Bacillus  nanus 

119.  Bacillus  ochraceus     [Zimmermann] 

120.  Bacillus  oleraceae 

121.  Bacillus  olfactorius 

122.  Oidium  lactis 

123.  Bacillus  oleae     [Schiff-Giorgini] 

124.  Cladothrix  odorifera 

125.  Cladothrix  dichotoma. 

126.  Bacillus  oxalatus 

127.  Bacterium  para-coli  gasformans  anindolicum    [Kayser] 

128.  Bacillus  parvus 

129.  Rhodobacillus  palustis 

130.  Bacillus  Petasites    [A.  Meyer  et  Gottheil] 

131.  Bacterium  Petroselini     [Bur chard] 

132.  Bacillus  prodigiosus    [Flugge] 

133.  Bacillus  lactis  proteolyticus     [Rullman] 

134.  Bacillus  plicatus 

135.  Bacterium  phytophtorum 

136.  Bacillus  proteus 

137.  Bacillus  pumilis     [A.  Meyer  et  Gottheil] 

138.  Bacillus  punctatus 

139.  Bacillus  fluorescens  putidus    [Flugge] 

140.  Bacillus  phosphorescens 

141.  Pseudomonas  pyocyanea 

142.  Bacterium  radiatum    [Kayser] 

143.  Pseudomonas  radicicola,  clover 

144.  Bacillus  ramosus  non  liquefaciens 

145.  Bacillus  rosaceus 

146.  Micrococcus  roseus    [Eisenberg] 

147.  Bacillus  of  ropy  milk 

148.  Micrococcus  rhodochrous 

149.  Bacillus  brunneus  mycoides  roseus 


36 

150.  Bacillus  capsulatus  roseus 

151.  Bacillus  ruber 

152.  Micrococcus  ruber 

153.  Bacillus  subtilis  var  ruber 

154.  Bacillus  ruber  Plymouth 

155.  Bacillus  rubidus 

156.  Spirillum  rubrum 

157.  Bacterium  rugosum     [Henrici] 

158.  Bacillus  ruber  of  Kiel 

159.  Spirillum  rugula 

160.  Bacterium  rubilum 

161.  Bacillus  ruminatus 

162.  Bacillus  rutilus 

163.  Bacillus  rutilensis 

164.  Spirillum  serpens 

165.  Bacillus  silvaticus     [Arthur  Meyer  et  Neide] 

166.  Bacillus  simplex     [A.  Meyer  et  Gottheil] 

167.  Vibrio  saprophilus 

168.  Micrococcus  sordidus 

169.  Bacillus  luteus  sporogenes     [Wood,  Smith  et  Baker] 

170.  Bacterium  der  sorbose 

171.  Bacillus  solanisaprus 

172.  Bacillus  sphaericus     [Arthur,  Meyer  et  Neide] 

173.  Staphlococcus  cereus  aureus 

174.  Staphlococcus  pyogenes  citreus 

175.  Staphlococcus  pyogenes  albus 

176.  Staphlococcus  pyogenes  aureus 

177.  Bacillus  ochraceus  subflavus 

178.  Bacterium  subflavum 

179.  Micrococcus  sulfur 

180.  Bacillus  subtilis     [Ehrenberg] 

181.  Bacterium  Stutzeri     [H .  Jensen] 

182.  Bacillus  synxanthus     [Cohn] 

183.  Bacterium  tremellioides     [Schottelius] 

184.  Bacillus  tumefaciens 

185.  Bacillus  tumescens 

186.  Bacillus  typhosus     [Eberth] 

187.  Bacillus  para- typhosus 

188.  Sarcina  ventriculi     [Goodsir] 

189.  Bacillus  violaceus     [Jordan] 


37 


190.  Azotobacter  vinelandii     [Lipman] 

191.  Micrococcus  viticulosus 

192.  Bacillus  proteus  viridis 

193.  Bacillus  aquatile  villos 

194.  Bacillus  vivax 

195.  Spirillum  volutans 

196.  Bacillus  proteus  vulgaris     [Hauser] 

197.  Bacterium  xanthochlorum 

198.  Bacillus  xylinum 

199.  Bacillus  proteus  Zenkeri     [Hauser] 

200.  Bacillus  Zopfii 

201.  Boden  I.     [Tsiklinsky-Sudpolar expedition  1903-5] 


1.  Bacterium  acetosum:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  considerably  diminished; 

Strong  formation  of  nitrite. 

2.  Bacterium  lactis  aerogenes:  good  growth;  vigorous  evolution  of  gas 

on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

3.  Bacillus  brassicae  acidae:   good  growth;   moderate  evolution  of  gas 

on  addition  of  acid;   nitrate  content  considerably  diminished; 

Weak  formation  of  nitrite. 

4.  Micrococcus  agilis:    good  growth;    no  evolution  of  gas  on  addition 

of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

5.  Bacillus  acidi  lactici:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

6.  Micrococcus  albidus:   moderate  growth;   slight  evolution  of  gas  on 

addition  of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

7.  Bacillus  amylovorus:    good  growth;    strong  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

8.  Bacillus  anthracis:   good  growth;   vigorous  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  on  nitrite. 

9.  Bacillus  pseudo-anthracis:    good  growth;    no  evolution  of  gas  on 

addition  of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

10.     Bacillus  anthracoides:   good  growth;   moderate  evolution  of  gas  on 
addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 


38 

11.  Bacterium  annulatum  A:    good  growth;    vigorous  evolution  of  gas 

on  addition  of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

12.  Bacterium  annulatum  B:    good  growth;    vigorous  evolution  of  gas 

on  addition  of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

13.  Bacillus  aquatile:   good  growth;   vigorous  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

14.  Bacillus  arborescens:   good  growth;   vigorous  evolution  of  gas  addi- 

tion of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

15.  Bacillus  argentinensis:  good  growth;  moderate  evolution  of  gas  on 

addition  of  acid;  nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

16.  Micrococcus  ascoformans:    fair  growth;    slight  evolution  of  gas  on 

addition  of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

17.  Bacillus  asterosporus:    scant  growth;    slight  evolution  of  gas  on 

addition  of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

18.  Bacterium  aurantiacus:   good  growth;   no  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

19.  Sarcina  aurantiaca:   scant  growth;   no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

20.  Bacillus  Baccarinii:  fair  growth;  slight  evolution  of  gas  on  addition 

of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

21.  Bacterium  beticolum:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;  nitrate  content  greatly  reduced; 

Moderate  formation  of  nitrite. 

22.  Micrococcus  brunneus:    moderate  growth;    slight  evolution  of  gas 

on  addition  of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

23.  Bacillus  budapestiensis:  good  growth;   no  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  very  slightly  diminished; 

Very  weak  formation  of  nitrite. 

24.  Bacillus  butyricus:    good  growth;    no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged' 

No  formation  of  nitrite. 

25.  Bacillus  candicans:  moderate  growth;   no  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

26.  Micrococcus  candicans:   moderate  growth;   no  evolution  of  gas  on 

addition  of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 


39 

27.  Monila  Candida:    fair  growth;    slight  evolution  of  gas  on  addition 

of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

28.  Bacillus  campestris:   good  growth;   no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

29.  Rhodobacillus  capsulatus:    moderate  growth;    no  evolution  of  gas 

on  addition  of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

30.  Bacillus  cereus:  good  growth;  vigorous  evolution  of  gas  on  addition 

of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

31.  Bacillus  cereulens:    good  growth;    moderate  evolution  of  gas  on 

addition  of  acid;  nitrate  content  appreciably  diminished; 

Weak  formation  of  nitrite. 

32.  Micrococcus  cereus:    moderate  growth;    no  evolution  of  gas  on 

addition  of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

33.  Micrococcus  carneus:  good  growth;  no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

34.  Micrococcus  cinnabareus:    fair  growth;    slight  evolution  of  gas  on 

addition  of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

35.  Bacillus  cloacae:   good  growth;   vigorous  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

36.  Micrococcus  citreus:   fair  growth;   no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

37.  Bacillus  constrictus:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

38.  Micrococcus  concentricus:  fair  growth;  no  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

39.  Bacillus  coli  commune:   good  growth;   vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

40.  Bacillus  coli-anaerogenes:   good  growth;    vigorous  evolution  of  gas 

on  addition  of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

41.  Bacillus  carotovorus:   fair  growth;  slight  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

42.  Bacillus  cyanogenes:   good  growth;   slight  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 


43.  Bacillus  cylindrosporus:    fair  growth;    slight  evolution  of  gas  on 

addition  of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

44.  Bacillus  creusus:   good  growth;   moderate  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  considerably  diminished; 

Moderate  formation  of  nitrite. 

45.  Bacillus  cyaneus:   good  growth;   no  evolution  of  gas  on  addition  of 

acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

46.  Bacterium  crysogloia:    moderate  growth;    no  evolution  of  gas  on 

addition  of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

47.  Bacillus  denitrificans:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

48.  Bacillus  dendroides:    fair  growth;    no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

49.  Pseudomonas  destructans:    moderate  growth;    fair  evolution  of  gas 

on  addition  of  acid;   nitrogen  content  slightly  diminished; 

Weak  formation  of  nitrite. 

50.  Bacillus  disciformans:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

51.  Bacillus  enteritidis:  good  growth;  vigorous  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

52.  Bacillus  esterigenes:    fair  growth;    no  evolution  of  gas  on  addition 

of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

53.  Bacillus  esterigenes  A:  good  growth;  no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

54.  Bacillus  esterigenes  D:  good  growth;  no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

55.  Bacterium  lactis  erythrogenes:    good  growth;    slight  evolution  of 

gas  on  addition  of  acid;    nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

56.  Bacillus  ethacinicus:    fair  growth;    moderate  evolution  of  gas  on 

addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

57.  Bacillus   ethaceto   succinicus:     good   growth;     moderate   evolution 

of  gas  on  addition  of  acid;  nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

58.  Bacillus  ferruginous:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 


41 

59.  Bacillus  faecalis  alcaligenes:    good  growth;    slight  evolution  of  gas 

on  addition  of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

60.  Sarcina  flava:    fair  growth;    slight  evolution  of  gas  on  addition  of 

acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

61.  Micrococcus  flavus:    moderate  growth;    slight  evolution  of  gas  on 

addition  of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

62.  Bacillus  flavidus:   moderate  growth;  weak  evolution  of  gas  on  addi- 

tion of  acid;   nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

63.  Bacterium  filiforme:   good  growth;   slight  evolution  of  gas  on  addi- 

tion of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

64.  Bacterium  filifaciens:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

65.  Bacillus  Fitzianus:  good  growth;  slight  evolution  of  gas  on  addition 

of  acid;   nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

66.  Bacillus  fluorescens  liquefaciens:    good  growth;    vigorous  evolution 

of  gas  on  addition  of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

67.  Bacillus  fluorescens  non-liquefaciens:   good  growth;   vigorous  evolu- 

tion of  gas  on  addition  of  acid;    nitrate  content  greatly  dimin- 
ished; Strong  formation  of  nitrite. 

68.  Bacillus  fluorescens  tenuis:    good  growth;    no  evolution  of  gas  on 

addition  of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

69.  Bacillus  Frostii:    good  growth;   slight  evolution  of  gas  on  addition 

of  acid;   nitrate  content  not  greatly  diminished. 

Weak  formation  of  nitrite. 

70.  Bacillus  fuchsinus:    moderate  growth;    no  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

71.  Sarcina  gasformans:    moderate  growth;    no   evolution  of  gas  on 

addition  of  acid;   nitrite  content  unchanged; 

No  formation  of  nitrite. 

72.  Bacillus  graviolens:   good  growth;   no  evolution  of  gas  on  addition 

of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

73.  Bacterium  aquatile  griseum:    good  growth;    vigorous  evolution  of 

gas  on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

74.  Micrococcus  grossus:   moderate  growth;   slight  evolution  of  gas  on 

addition  of  acid;   nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 


42 

75.  Bacterium  Hartlebi:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

76.  Bacillus  Havaniensis:   good  growth;   moderate  evolution  of  gas  on 

addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

77.  Bacillus  herbicoli  aureus:  moderate  growth;   slight  evolution  of  gas 

on  addition  of  acid;  nitrate  content  diminished; 

Weak  formation  of  nitrite. 

78.  Bacillus  helvolus:   good  growth;   moderate  evolution  of  gas  on  ad- 

dition of  acid;  nitrate  content  diminished; 

Fair  formation  of  nitrite. 

79.  Bacillus  Hoagii:  moderate  growth;  fair  evolution  of  gas  on  addition 

of  acid;  nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

80.  Bacillus  hyponitrous:  scant  growth;  no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

81.  Bacillus  immobile:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

82.  Bacillus  indicus:    good  growth;   slight  evolution  of  gas  on  addition 

of  acid;   nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

83.  Bacillus  indigoferus:    good  growth;    moderate  evolution  of  gas  on 

addition  of  acid;   nitrate  content  slightly  diminished; 

Moderate  formation  of  nitrite. 

84.  Bacillus  irritans:    good  growth;   moderate  evolution  of  gas  on  ad- 

dition of  acid;  nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

85.  Bacillus  ivilans:    moderate  growth;    slight  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

86.  Bacillus  jasminocyaneus:    good  growth;    vigorous  evolution  of  gas 

on  addition  of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

87.  Bacillus  juglandis:    good  growth;    no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

88.  Bacillus  kiliensis:    moderate  growth;    no  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

89.  Bacillus  lactis:   good  growth;   vigorous  evolution  of  gas  on  addition 

of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

90.  Bacillus  lactorubefaciens:    good  growth;   vigorous  evolution  of  gas 

on  addition  of  acid;  nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 


43 

91.  Bacillus  lateritia:   good  growth;  slight  evolution  of  gas  on  addition 

of  acid;  nitrate  content  almost  unchanged; 

Very  weak  formation  of  nitrite. 

92.  Bacillus  levans:   moderate  growth;   no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

93.  Bacillus  lactis  amari  liquefaciens:   good  growth;   slight  evolution  of 

gas  on  addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

94.  Bacillus  liodermos:    good  growth;    moderate  evolution  of  gas  on 

addition  of  acid;   nitrate  content  considerably  diminished; 

Moderate  formation  of  nitrite. 

95.  Bacillus  limosus:    moderate  growth;    slight  evolution  of  gas  on 

addition  of  acid;   nitrate  content  not  materially  changed; 

Weak  formation  of  nitrite. 

96.  Sarcina  liquefaciens:    fair  growth;   no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

97.  Bacillus  liquefaciens:   good  growth;  no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

98.  Bacillus  lactis  niger:    good  growth;    moderate  evolution  of  gas  on 

addition  of  acid;   nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

99.  Bacillus  liquefaciens  niger:   good  growth;  moderate  evolution  of  gas 

on  addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

100.  Bacillus  loxosus:    good  growth;    vigorous  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

101.  Bacterium    aquatile    gasformans    non-liquefaciens:     good    growth; 

vigorous  evolution  of  gas  on  addition  of  acid;    nitrate  content 
greatly  diminished; 

Strong  formation  of  nitrite. 

102.  Micrococcus  luteus:    fair  growth;    no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

103.  Sarcina  lutea:   moderate  growth;  no  evolution  of  gas  on  addition  of 

acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

104.  Streptococcus  luteus  liquefaciens:   good  growth;  no  evolution  of  gas 

on  addition  of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

105.  Bacillus  maidis:    scant  growth;  no  evolution  of  gas  on  addition  of 

acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 


44 

106.  Bacillus  melonis:    good  growth;  vigorous  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

107.  Bacillus  mesentericus  fuscus:    good  growth;    vigorous  evolution  of 

gas  on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

108.  Bacillus  mesentericus  niger:   good  growth;  vigorous  evolution  of  gas 

on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

109.  Bacillus  mesentericus  ruber:    good  growth;  vigorous  evolution  of 

gas  on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

110.  Bacillus  mesentericus  vulgatus:   good  growth;    slight  evolution  of 

gas  on  addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

111.  Bacillus  megatherium:    good  growth;   no  evolution  of  gas  on  addi- 

tion of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

112.  Bacillus  miniaceus:     good   growth;   vigorous  evolution   of   gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

113.  Bacillus  proteus  mirabilis:    good  growth;  no  evolution  of  gas  on 

addition  of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

114.  Sarcina  mobilis:   fair  growth;   slight  evolution  of  gas  on  addition  of 

acid;  nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

115.  Moeller's  grass  bacillus,  Mist:   good  growth;  moderate  evolution  of 

gas  on  addition  of  acid;   nitrate  content  slightly  diminished; 

Moderate  formation  of  nitrite. 

116.  Bacterium  muris:   fair  growth;    no  evolution  of  gas  on  addition  of 

acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

117.  Bacillus   mycoides:    good   growth;    vigorous   evolution   of   gas   on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

118.  Bacillus  nanus:   good  growth;  vigorous  evolution  of  gas  on  addition 

of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

119.  Bacillus  ochraceus:    good  growth;  no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

120.  Bacillus  oleraceae:    moderate  growth;  fair  evolution  of  gas  on  ad- 

dition of  acid;    nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

121.  Bacillus  olfactorius:    good  growth;  vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 


45 

122.  Oidium  lactis:    good  growth;   no  evolution  of  gas  on  addition  of 

acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

123.  Bacillus  oleae:    good  growth;   vigorous  evolution  of  gas  on  addition 

of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

124.  Cladothrix  odorifera:   good  growth;  no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

125.  Cladothrix  dichotoma:   good  growth;   no  evolution  of   gas  on  ad- 

dition of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

126.  Bacillus  oxalatus:   good  growth;  strong  evolution  of  gas  on  addition 

of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

127.  Bacterium  paracoli  gasformans  anindolicum:    good  growth;  moder- 

ate evolution  of  gas  on  addition  of  acid;   nitrate  content  slightly 
diminished; 

Moderate  formation  of  nitrite. 

128.  Bacillus  parvus:   good  growth;   vigorous  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

129.  Rhodobacillus  palustis:    good  growth;   vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

130.  Bacillus  Petasites:    good  growth;  no  evolution  of  gas  on  addition 

of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

131.  Bacterium  Petroselini:    good  growth;   moderate  evolution  of  gas  on 

addition  of  acid;    nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

132.  Bacillus  prodigiosus:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

133.  Bacillus  lactis  proteolyticus:   good  growth;    vigorous  evolution  of 

gas  on  addition  of  acid;    nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

134.  Bacillus   plicatus:   moderate   growth;    slight   evolution   of   gas   on 

addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

135.  Bacterium   phytophtorum :   fair  growth;    no   evolution   of   gas   on 

addition  of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

136.  Bacillus  proteus:    good  growth;    moderate  evolution  of  gas  on  ad- 

dition of  acid;    nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

137.  Bacillus  pumilus:    good  growth;   no  evolution  of  gas  on  addition  of 

acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 


46 

138.  Bacillus  punctatus:    good  growth;   vigorous  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

139.  Bacillus  fluorescens  putidus:   moderate  growth;   no  evolution  of  gas 

on  addition  of  acid;   nitrate  content  undiminished; 

No  formation  of  nitrite. 

140.  Bacillus  phosphorescens:   good  growth;  vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

141.  Pseudomonas  pyocyanea:    good  growth;    vigorous  evolution  of  gas 

on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

142.  Bacterium  radiatum:   good  growth;  no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

143.  Pseudomonas  radicicola,  clover:    good  growth;    vigorous  evolution 

of  gas  on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

144.  Bacillus  ramosus  non  liquefaciens:    good  growth;   no  evolution  of 

gas  on  addition  of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

145.  Bacillus  rosaceus:   good  growth;   no  evolution  of  gas  on  addition  of 

acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

146.  Micrococcus  roseus:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

147.  Bacillus  of  ropy  milk:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

148.  Micrococcus  rhodochrous:  moderate  growth;  slight  evolution  of  gas 

on  addition  of  acid;   nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

149.  Bacillus  brunneus  mycoides  roseus:  good  growth;  vigorous  evolution 

of  gas  on  addition  of  acid;    nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

150.  Bacillus  capsulatus  roseus:   good  growth;   vigorous  evolution  of  gas 

on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

151.  Bacillus  ruber:   moderate  growth;  fair  evolution  of  gas  on  addition 

of  acid;  nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

152.  Micrococcus  ruber:    moderate  growth;    fair  evolution  of  gas  on 

addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

153.  Bacillus  subtilis  var  ruber:   good  growth;  vigorous  evolution  of  gas 

on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 


47 

154.  Bacillus  ruber  Plymouth:    good  growth;    vigorous  evolution  of  gas 

on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

155.  Bacillus  rubidus:    good  growth;   no  evolution  of  gas  on  addition  of 

acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

156.  Spirillum  rubrum:    moderate  growth;    no  evolution  of  gas  on  ad- 

dition of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

157.  Bacterium  rugosum:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

158.  Bacillus  ruber  of  Kiel:    good  growth;   vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

159.  Spirillum  Rugala:  moderate  growth;  no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

160.  Bacterium  rubilum:   fair  growth;  no  evolution  of  gas  on  addition  of 

acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

161.  Bacillus  ruminatus:    good  growth;   no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

162.  Bacillus  rutilus:    good  growth;    moderate  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

163.  Bacillus  rutilensis:     fair   growth;    moderate   evolution   of  gas   on 

addition  of  acid;   nitrate  content  slighlty  diminished; 

Weak  formation  of  nitrite. 

164.  Spirillum  serpens:    moderate  growth;    no  evolution  of  gas  on  the 

addition  of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

165.  Bacillus  silvaticus:     good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

166.  Bacillus  simplex:    good  growth;    vigorous  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  greatly  diminished ,v 

Strong  formation  of  nitrite. 

167.  Vibrio  saprophilus:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

168.  Micrococcus  sordidus:   moderate  growth;  slight  evolution  of  gas  on 

addition  of  acid;   nitrate  content  diminished; 

Weak  formation  of  nitrite. 

169.  Bacillus  luteus  sporogenes:    good  growth;    no  evolution  of  gas  on 

addition  of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 


48 

170.  Bacterium  der  sorbose:    good  growth;   vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

171.  Bacillus  solanisparus:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

172.  Bacillus  sphaericus:   good  growth;  no  evolution  of  gas  on  addition  of 

acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

173.  Staphlococcus  cereus  aureus:    good  growth;    moderate  evolution  of 

gas  on  addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

174.  Staphlococcus  pyogenes  citreus:    good  growth;    no  evolution  of  gas 

on  addition  of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

175.  Staphlococcus  pyogenes  albus:   good  growth;  moderate  evolution  of 

gas  on  addition  of  acid;   nitrate  content  slightly  reduced; 

Weak  formation  of  nitrite. 

176.  Staphlococcus  pyogenes  aureus:    good  growth;   fair  evolution  of  gas 

on  addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

177.  Bacillus  ochraceus  subflavus:    good  growth;    vigorous  evolution  of 

gas  on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  furmation  of  nitrite. 

178.  Bacterium  subflavum:   fair  growth;   no  evolution  of  gas  on  addition 

of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

179.  Micrococcus  sulfur:    moderate  growth;    no   evolution   of   gas   on 

addition  of  acid;   nitrate  content  unchanged; 

No  formation  of  nitrite. 

180.  Bacillus  subtilis:    good  growth;    moderate  evolution  of  gas  on  ad- 

dition of  acid;    nitrate  content  slightly  diminished; 

Weak  furmation  of  nitrite. 

181.  Bacterium  Stutzeri:    good  growth;    strong  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

182.  Bacillus  synxanthus:    good  growth;    moderate  evolution  of  gas  on 

addition  of  acid;   nitrate  content  moderately  diminished; 

Weak  formation  of  nitrite. 

183.  Bacterium  tremellioides:   good  growth;  vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

184.  Bacillus  tumefaciens:    fair  growth;   no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

185.  Bacillus  tumescens:    good  growth;   no  evolution  of  gas  on  addition 

of  acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 


49 

186.  Bacillus  typhosus:    good  growth;    vigorous  evolution  of  gas  on 

addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

187.  Bacillus  para-typhosus:    moderate  growth;    slight  evolution  of  gas 

on  addition  of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

188.  Sarcina  ventriculi:    fair  growth;    moderate  evolution  of  gas  on 

addition  of  acid;   nitrate  content  considerably  diminished; 

Moderate  formation  of  nitrite. 

189.  Bacillus  violaceus:    moderate  growth;   fair  evolution  of  gas  on  .ad- 

dition of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

190.  Azotobacter  vinelandi:   good  growth;  moderate  evolution  of  gas  on 

addition  of  acid;   nitrate  content  considerably  diminished; 

Weak  formation  of  nitrite. 

191.  Micrococcus  viticulosus:    moderate  growth;   slight  evolution  of  gas 

on  addition  of  acid;  nitrate  content  not  greatly  diminished; 

Weak  formation  of  nitrite. 

192.  Bacillus  viridis:    good  growth;    vigorous  evolution  of  gas  on  ad- 

dition of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

193.  Bacillus  aquatile  villos:    moderate  growth;    slight  evolution  of  gas 

on  addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

194.  Bacillus  vivax:    fair  growth;    no  evolution  of  gas  on  addition  of 

acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

195.  Spirillum  volutans:    good  growth;    moderate  evolution  of  gas  on 

addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

196.  Bacillus  proteus  vulgaris:    good  growth;   vigorous  evolution  of  gas 

on  addition  of  acid;   nitrate  content  greatly  diminished; 

Strong  formation  of  nitrite. 

197.  Bacterium  xanthochlorum:    good  growth;    moderate  evolution  of 

gas  on  addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

198.  Bacillus  xylinum:    good  growth;    moderate  evolution  of  gas  on 

addition  of  acid;   nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

199.  Bacillus  proteus  Zenkeri:    good  growth;   moderate  evolution  of  gas 

on  addition  of  acid:  nitrate  content  slightly  diminished; 

Weak  formation  of  nitrite. 

200.  Bacillus  Zopfii:    scant  growth;    no  evolution  of  gas  on  addition  of 

acid;  nitrate  content  unchanged; 

No  formation  of  nitrite. 

201.  Boden  I   (Tsiklinsky-Sudpolar  expedition,  1903-5):    good  growth; 

vigorous  evolution  of  gas  on  addition  of  acid;    nitrate  content 
greatly  diminished; 

Strong  formation  of  nitrite. 


50 

THE  FOLLOWING  ORGANISMS  REDUCED  NITRATE 

TO  NITRITE 

1.  Bacterium  acetosum;  2.  Bacterium  lactis  aerogenes; 
3.  Bacillus  brassicae  acidae;  5.  Bacillus  acidi  lactici;  6.  Mi- 
crococcus  albidus;  7.  Bacillus  amylovorus;  8.  Bacillus  anth- 
racis;  10.  Bacillus  anthracoides;  11.  Bacterium  annulatumA; 
12.  Bacterium  annulatum  B;  13.  Bacillus  aquatile;  14.  Bacil- 
lus arborescens;  15.  Bacillus  argentinensis;  16.  Micrococcus 
ascoformans;  17.  Bacillus  asterosporus;  20.  Bacillus  baccar- 
inii;  21.  Bacterium  beticolum;  22.  Micrococcus  brunneus; 
23.  Bacillus  budapestiensis;  27.  Monila  Candida;  30.  Bacillus 
cereus;  31.  Bacillus  cereulens;  34.  Micrococcus  cinnabareus; 
35.  Bacillus  cloacae;  37.  Bacillus  constrictus;  39.  Bacillus 
coli  commune;  40.  Bacillus  coli-anaerogenes;  41.  Bacillus 
carotovorus;  42.  Bacillus  cyanogenes;  43.  Bacillus  cylin- 
drosporus;  44.  Bacillus  creusus;  47.  Bacillus  denitrificans; 
49.  Pseudomonas  destructans;  50.  Bacillus  disciformans; 
51.  Bacillus  enteritidis;  55.  Bacterium  lactis  erythrogenes; 
56.  Bacillus  ethacinicus;  57.  Bacillus  ethaceto  succinus;  58. 
Bacillus  ferruginous;  59.  Bacillus  faecalis  alcaligenes;  60. 
Sarcina  flava;  61.  Micrococcus  flavus;  62.  Bacillus  flavidus; 
63.  Bacterium  filiforme;  64.  Bacterium  filifaciens;  65.  Bacil- 
lus Fitzianus;  66.  Bacillus  fluorescens  liquefaciens;  67. 
Bacillus  fluorescens  non  liquefaciens;  69.  Bacillus  Frostii; 
73.  Bacillus  aquatile  griseum;  74.  Micrococcus  grossus;  75. 
Bacterium  Hartlebi;  76.  Bacillus  Havaniensis;  77.  Bacillus 
herbicoli  aureus;  78.  Bacillus  helvolus;  79.  Bacillus  Hoagii; 
81.  Bacillus  immobile;  82.  Bacillus  indicus;  83.  Bacillus 
indigoferus;  84.  Bacillus  irritans;  85.  Bacillus  ivilans;  86. 
Bacillus  jasminocyaneus;  89.  Bacillus  lactis;  90.  Bacillus 
lactorubefaciens;  91.  Bacillus  lateritia;  93.  Bacillus  lactis 
amari  liquefaciens;  94.  Bacillus  liodermos;  95.  Bacillus 
limosus;  98.  Bacillus  lactis  niger;  99.  Bacillus  liquefaciens 
niger;  100.  Bacillus  loxosus;  101.  Bacterium  aquatile  gas- 
formans  non  liquefaciens;  106.  Bacillus  melonis;  107.  Bacillus 
mesentericus  fuscus;  108.  Bacillus  mesentericus  niger;  109. 
Bacillus  mesentericus  ruber;  110.  Bacillus  mesentericus 
vulgatus;  112.  Bacillus  miniaceus;  114.  Sarcina  mobilis, 
115.  Moeller's  grass  bacillus,  Mist.;  117.  Bacillus  mycoides; 


51 

118.  Bacillus  nanus;  120.  Bacillus  oleraceae;  121.  Bacillus 
olfactorius;  123.  Bacillus  oleae;  126.  Bacillus  oxalatus;  127. 
Bacterium  paracoli  gasformans  anindolicum;  128.  Bacillus 
parvus;  129.  Rhodobacillus  palustis;  131.  Bacterium  Petro- 
selini;  132.  Bacillus  prodigiosus;  133.  Bacillus  lactis  proteo- 
lyticus;  134.  Bacillus  plicatus;  136.  Bacillus  proteus;  138. 
Bacillus  punctatus;  140.  Bacillus  phosphorescens;  141. 
Pseudomanaspyocyanea;  143.  Pseudomonas  radicicola  clover; 
146.  Micrococcus  roseus;  147.  Bacillus  of  ropy  milk;  148. 
Micrococcus  rhodochrous;  149.  Bacillus  brunneus  mycoides 
roseus;  150.  Bacillus  capsulatus  roseus;  151.  Bacillus  ruber; 
152.  Micrococcus  ruber;  153.  Bacillus  subtilis  var  ruber;  154. 
Bacillus  ruber  Plymouth;  157.  Bacterium  rugosum;  158. 
Bacillus  ruber  of  Kiel;  162.  Bacillus  rutilus;  163.  Bacillus 
rutilensis;  165.  Bacillus  silvaticus;  166.  Bacillus  simplex; 
167.  Vibrio  saprophilus;  168.  Micrococcus  sordidus;  170. 
Bacterium  der  sorbose;  171.  Bacillus  solanisparus;  173. 
Staphlococcus  cereus  aureus;  175.  Staphlococcus  pyogenes 
albus;  176.  Staphlococcus  pyogenes  aureus;  177.  Bacillus 
ochraceus  subflavus;  180.  Bacillus  subtilis;  181.  Bacterium 
Stutzeri;  182.  Bacillus  synxanthus;  183.  Bacterium  tremel- 
lioides;  186.  Bacillus  typhosus;  187.  Bacillus  paratyphosus; 
188.  Sarcina  ventriculi;  189.  Bacillus  violaceus;  190.  Azoto- 
bacter  vinelandii;  191.  Micrococcus  viticulosus;  192.  Bacillus 
proteus  viridis;  193.  Bacillus  aquatile  villos;  195.  Spirillum 
volutans;  196.  Bacillus  proteus  vulgaris;  197.  Bacterium 
xanthochlorum;  198.  Bacillus  xylinum;  199.  Bacillus  proteus 
Zenkeri;  201.  Boden  I.  (Tsiklinsky  Siidpolarexpedition 
1903—5). 

THE  FOLLOWING  ORGANISMS  DID  NOT  REDUCE 
NITRATE  TO  NITRITE 

4.  Micrococcus  agilis;  9.  Bacillus  pseudo-anthracis;  18. 
Bacterium  aurantiacus;  19.  Sarcina  aurantiaca;  24.  Bacillus 
butyricus;  25.  Bacillus  candicans;  26.  Micrococcus  candicans; 
28.  Bacillus  campestris;  29.  Rhodobacillus  capsulatus;  32. 
Micrococcus  cereus;  33.  Micrococcus  carneus;  36.  Micrococ- 
cus citreus;  38.  Micrococcus  concentricus;  45.  Bacillus 
cyaneus;  46.  Bacterium  crysogloia;  48.  Bacillus  dendroides; 


52 

51.  Bacillus  esterigenes;  53.  Bacillus  esterigenes  A;  54.  Bacil- 
lus esterigenes  D;  68.  Bacillus  fluorescens  tenuis;  70.  Bacillus 
fuchsinus;  71.  Sarcina  gasformans;  72.  Bacillus  graviolens; 
80.  Bacillus  hyponitrous;  87.  Bacillus  juglandis;  88.  Bacillus 
kiliensis;  92.  Bacillus  levans;  96.  Sarcina  liquefaciens;  97. 
Bacillus  liquefaciens;  102.  Micrococcus  luteus;  103.  Sarcina 
lutea;  104.  Streptococcus  luteus  liquefaciens;  105.  Bacillus 
maidis;  111.  Bacillus  megatherium;  113.  Bacillus  proteus 
mirabilis;  116.  Bacterium  muris;  119.  Bacillus  ochraceus; 
122.  Oidium  lactis;  124.  Cladothrix  odorifera;  125.  Cladothrix 
dichotoma;  130.  Bacillus  Petasites;  135.  Bacterium  phyto- 
phtorum;  137.  Bacillus  pumilis;  139.  Bacillus  fluorescens 
putidus;  142.  Bacterium  radiatum;  144.  Bacillus  ramosus 
non  liquefaciens;  145.  Bacillus  rosaceus;  155.  Bacillus  rubidus; 
156.  Spirillum  rubrum;  159.  Spirillum  Rugula;  160.  Bac- 
terium rubilum;  161.  Bacillus  ruminatus;  164.  Spirillum 
serpens;  169.  Bacterium  luteus  sporogenes;  172.  Bacillus 
sphaericus;  174.  Staphlococcus  pyogenes  citreus;  178.  Bac- 
terium subflavum;  179.  Micrococcus  sulfur;  184.  Bacillus 
tumefaciens;  185.  Bacillus  tumescens;  194.  Bacillus  vivax; 
200.  Bacillus  Zopfii. 

Of  the  201  organisms  under  consideration  139,  or  69.1%, 
reduced  nitrate  to  nitrite,  and  62,  or  30.9%,  did  not  effect 
this  reduction.  Those  organisms  which  produce  green  pig- 
ment almost  invariably  reduce  nitrate  to  free  nitrogen.  This 
reduction  takes  place  very  rapidly,  after  forty-eight  hours 
no  nitrite  remains  in  solutions  of  small  concentration.  It  is 
impossible  to  declare  from  the  vigor  of  the  growth  of  the 
organism  respecting  its  ability  to  effect  the  reduction  of  nitrate 
to  nitrite.  However,  those  bacteria  which  failed  to  perform 
such  reduction  were  commonly  found  among  those  whose 
growth  was  slow  and  at  best  feeble. 

Many  organisms  were  inoculated  into  Giltay's  medium  in 
the  hope  that  it  would  prove  available  for  reduction  experi- 
ments. This  proved  to  be  the  case  with  soil  inoculation,  but 
in  pure  culture  the  slow  growth  of  all  and  the  refusal  of  many 
bacteria  to  develop  in  this  synthetic  medium,  did  not  prove 
encouraging.  Calcium  glycerophosphate  and  calcium  lac- 
tophosphate  were  also  tried  in  this  connection.  These  com- 
pounds proved  to  be  unstable  in  solution  and  were  difficult 


\ 


53 

to  sterilize  intact.  Nothing  was  found  superior  to  peptone 
although  this  medium  in  the  absence  of  mineral  salts  and  car- 
bonaceous material  is  far  from  the  optimum  requirement  of 
most  bacteria. 

DENITRIFICATION 

It  must  be  evident  that  of  all  the  organisms  which  have 
been  studied,  comparatively  few  reduce  nitrite  to  free  nitrogen 
in  pure  culture.  Of  the  seventy  soils  under  consideration,  all 
of  those  which  would  be  considered  as  suitable  for  crop  pro- 
duction reduced  nitrite  to  free  nitrogen  in  a  very  short  period 
of  time.  It  must  therefore  be  concluded  that  either  the  very 
few  species  of  bacteria  which  effect  such  reduction  are  univer- 
sally distributed,  or  that  those  organisms  which  will  not  per- 
form this  function  in  pure  culture  will  work  in  symbiosis  to 
effect  this  end.  The  latter  phenomenon  has  been  established 
with  reference  to  a  few  organisms  and  will  doubtless  be  extended 
to  include  a  great  variety.  The  operation  of  this  function 
is  not  confined  to  nitrogenous  media  but  is  performed  with 
equal  vigor  in  Ashby's  medium  in  which  either  glucose  or  man- 
nite  are  employed.  The  reduction  seemingly  taking  place  a 
little  slower  in  the  case  of  glucose.  In  simple  soil  infusion  of 
considerable  concentration  I  have  been  unable  to  detect  the 
slightest  evidence  of  reduction  of  nitrates  or  nitrites  on  addi- 
tion of  these  salts.  That  these  reductions  are  effected  wholly 
according  to  the  nascent  hydrogen  theory  seems  improbable. 
By  soil  inoculation  of  the  media  I  have  been  unable  to  reduce 
sulphates  to  hydrogen  sulphide  except  in  a  very  few  instances, 
while  with  sewage  sludge  no  difficulty  is  experienced.  The 
phosphates  also  seem  refractory.  A  selective  action  involving 
energy,  nutrition,  etc.,  may  be  concerned. 

But  eight  species  of  bacteria  are  commonly  cited  as  re- 
ducing nitrites  to  free  nitrogen  in  pure  culture:  Bacterium 
centropunctatum,  (H.  Jensen),  Bacterium  filifaciens  (H. 
Jensen),  Bacterium  Hartlebi  (H.  Jensen),  Bacterium  nitro- 
vorum  (H.  Jensen),  Pseudomonas  pyocyanea  (Migula),  Bacil- 
lus denitrificans  (H.  Jensen)  and  probably  two  members  of 
the  Fluorescens  group:  Bacillus  fluorescens  and  Bacillus 
fluorescens  liquefaciens.  Maassen  observed  that  Bacillus 
praepollens  broke  down  nitrates  to  free  nitrogen  only  in  sym- 


54 

biosis  with  other  bacteria,  and  that  nitrites  were  not  reduced 
to  free  nitrogen  except  in  harmony  with  organisms  which  re- 
duced nitrates  to  nitrites.  He  discovered  that  the  following 
organisms,  in  cooperation  with  Bacillus  praepollens,  would 
effect  this  reduction:  "B.  acidi  lactici,  B.  capsulatus,  B.  cre- 
moides,  B.  cuniculicida  mobilis,  B.  diphtheria  columbarum, 
B.  enteritidis  Gartneri,  B.  from  lean  meat,  B.  indigonaceus, 
B.  mycoides,  B.  mesentericus  ruber,  B.  mesentericus  Flugge 
I,  III  and  VII,  B.  mustelae  septicus,  B.  miniaceus,  B.  pro- 
digiosus,  B.  pneumoniae,  B.  proteus  mirabilis,  B.  proteus 
vulgaris,  B.  psitticosis,  B.  rhinoscleromatis,  B.  ruber  of  Kiel, 
B.  ruber  plymouth,  B.  ruber  purpureus,  B.  suipestifer,  B. 
Hog-cholera  (Salmon),  B.  swine  plague,  B.  typhi-abdominalis, 
B.  typhi-murium,  B.  violaceus,  B.  coli  commune  I,  II,  III,  IV, 
B.  lactis  aerogenes,  B.  phosphorescens,  M.  candicans,  Staph- 
lococcus  pyogenes  albus,  Staphlococcus  pyogenes  aureus, 
Sarcina  flava  II,  Vibrio  Blankenese,  Vibrio  Mottlau  II,  Vibrio 
tyrogenes  Deneke." 

An  attempt  was  made  to  isolate  the  organisms  from  the 
soil,  which  perform  the  function  of  reducing  nitrates  to  free 
nitrogen.  As  far  as  possible  all  the  organisms  were  isolated 
from  soil  number  9.  None  of  these  bacteria  reduced  nitrates 
to  free  nitrogen  in  pure  culture.  Of  the  fifteen  different  species 
thus  isolated  various  combinations  were  made  in  the  hope  of 
discovering  a  symbiotic  relation,  but  of  the  many  combina- 
tions thus  effected  in  no  instance  did  I  succeed  in  securing  the 
desired  result.  After  fishing  the  different  colonies  from  a 
great  number  of  plates,  the  agar  in  several  was  carefully  rolled 
together  with  a  sterile  spatula  and  introduced  into  a  medium 
prepared  for  the  reduction  of  nitrates,  those  plates  which  con- 
tained the  greater  number  of  colonies  invariably  reduced  the 
nitrate  to  free  nitrogen,  but  in  some  of  the  plates  on  which 
were  few  colonies  no  reduction  took  place. 

I  am  under  obligations  to  Dr.  A.  F.  MacLeod,  Assistant 
Professor  of  Physical  Chemistry  in  Beloit  College,  Beloit, 
Wisconsin,  for  valuable  suggestions  and  assistance;  and  like- 
wise to  Dr.  H.  H.  Waite,  Professor  of  Bacteriology  and  Patho- 
logy in  the  University  of  Nebraska,  for  valuable  suggestions 
and  assistance. 


28801*; 


MJ 


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